Methods for predicting response of triple-negative breast cancer to therapy

ABSTRACT

The present invention provides compositions and methods for detecting the expression and/or activation levels of components of signal transduction pathways in tumor cells such as triple-negative metastatic breast tumor cells. Information on the expression and/or activation levels of components of signal transduction pathways derived from use of the present invention can be used for cancer diagnosis, prognosis, and in the design of cancer treatments.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of PCT/US2011/021026, filed Jan. 12,2011, which application claims priority to U.S. Provisional ApplicationNo. 61/294,433, filed Jan. 12, 2010, U.S. Provisional Application No.61/325,624, filed Apr. 19, 2010, U.S. Provisional Application No.61/328,602, filed Apr. 27, 2010, and U.S. Provisional Application No.61/351,838, filed Jun. 4, 2010, the disclosures of which are hereinincorporated by reference in their entirety for all purposes.

BACKGROUND OF THE INVENTION

The process of signal transduction in cells is responsible for a varietyof biological functions including cell division and death, metabolism,immune cell activation, neurotransmission, and sensory perception toname but a few. Accordingly, derangements in normal signal transductionin cells can lead to a number of disease states such as diabetes, heartdisease, autoimmunity, and cancer.

One well characterized signal transduction pathway is the MAP kinasepathway, which is responsible for transducing the signal from epidermalgrowth factor (EGF) to the promotion of cell proliferation in cells(see, FIG. 1 of PCT Publication No. WO2009/108637, the disclosure ofwhich is herein incorporated by reference in its entirety for allpurposes). EGF binds to a transmembrane receptor-linked tyrosine kinase,the epidermal growth factor receptor (EGFR), which is activated by thebinding of EGF. The binding of EGF to EGFR activates the tyrosine kinaseactivity of the cytoplasmic domain of the receptor. One consequence ofthis kinase activation is the autophosphorylation of EGFR on tyrosineresidues. The phosphorylated tyrosine residues on the activated EGFRprovide a docking site for the binding of SH2 domain containing adaptorproteins such as GRB2. In its function as an adaptor, GRB2 further bindsto a guanine nucleotide exchange factor, SOS, by way of an SH3 domain onGRB2. The formation of the complex of EGFR-GRB2-SOS leads to SOSactivation of a guanine nucleotide exchange factor that promotes theremoval of GDP from Ras. Upon removal of GDP, Ras binds GTP and becomesactivated.

Following activation, Ras binds to and activates the protein kinaseactivity of RAF kinase, a serine/threonine-specific protein kinase. Whatfollows is the activation of a protein kinase cascade that leads to cellproliferation. In outline, RAF kinase then phosphorylates and activatesMEK, another serine/threonine kinase. Activated MEK phosphorylates andactivates mitogen-activated protein kinase (MAPK). Among the targets forfurther phosphorylation by MAPK are 40S ribosomal protein S6 kinase(RSK). The phosphorylation of RSK by MAPK results in activation of RSK,which in turn phosphorylates ribosomal protein S6. Another known targetof MAPK is the proto-oncogene, c-Myc, a gene important for cellproliferation, which is mutated in a variety of cancers. MAPK alsophosphorylates and activates another protein kinase, MNK, which in turnphosphorylates the transcription factor, CREB. Indirectly, MAPK alsoregulates the transcription of the Fos gene, which encodes yet anothertranscription factor involved in cell proliferation. By altering thelevels and activities of such transcription factors, MAPK transduces theoriginal extracellular signal from EGF into altered transcription ofgenes that are important for cell cycle progression.

Given the central role that signal transduction pathways play in cellgrowth, it is not surprising that many cancers arise as a result ofmutations and other alterations in signal transduction components thatresult in aberrant activation of cell proliferation pathways. Forexample, overexpression or hyperactivity of EGFR has been associatedwith a number of cancers, including glioblastoma multiforme, coloncancer, and lung cancer. This has prompted the development of anticancertherapeutics directed against EGFR, including gefitinib and erlotinibfor lung cancer, and cetuximab for colon cancer.

Cetuximab is an example of a monoclonal antibody inhibitor, which bindsto the extracellular ligand-binding domain of EGFR, thus preventing thebinding of ligands which activate the EGFR tyrosine kinase. In contrast,gefitinib and erlotinib are small molecules which inhibit theintracellularly-located EGFR tyrosine kinase. In the absence of kinaseactivity, EGFR is unable to undergo autophosphorylation at tyrosineresidues, which is a prerequisite for binding of downstream adaptorproteins, such as GRB2. By halting the signaling cascade in cells thatrely on this pathway for growth, tumor proliferation and migration isdiminished.

Additionally, other studies have shown that about 70% of human melanomasand a smaller fraction of other tumors have a point mutation (V599E) inthe Raf gene which leads to persistent activation of the MAPK pathway(see, e.g., Davies et al., Nature, 417:949-954 (2002)). Such resultssuggest that mutations in particular signal transduction pathways may becharacteristic of particular types of tumors and that such specific,altered signal transduction pathways may be a promising target forchemotherapeutic intervention.

Given that different cancer treatments, particularly cancerchemotherapy, may function either directly or indirectly by means ofeither blocking or activating cellular signal transduction pathways thatare involved in cell proliferation or death, respectively, the activityof a given signal transduction pathway in a particular form of cancermay serve as a good indicator of the efficacy of various cancertreatments. Accordingly, in addition to fulfilling other needs, thepresent invention provides methods for predicting and evaluating theeffectiveness of potential anticancer therapies for an individualpatient. As such, the present invention provides methods for assisting aphysician in selecting a suitable cancer therapy at the right dose andat the right time for every patient.

BRIEF SUMMARY OF THE INVENTION

The present invention provides compositions and methods for detectingthe status (e.g., expression and/or activation levels) of components ofsignal transduction pathways in tumor cells (e.g., triple-negativebreast tumor cells). Information on the expression and/or activationstates of components of signal transduction pathways derived frompractice of the present invention can be used for cancer diagnosis,prognosis, and in the design of cancer treatments.

In particular aspects, the present invention provides molecular markers(biomarkers) that enable the determination or prediction of whether aparticular cancer can respond or is likely to respond favorably to oneor more anticancer drugs such as, e.g., a combination of bevacizumab(Avastin®), carboplatin, and paclitaxel (e.g., Abraxane® or nabP)(“triplet therapy”). As described herein, it has been surprisingly foundthat biomarkers such as VEGFR2, c-KIT, HER1, and IGF-1R are particularlyuseful in determining or predicting the sensitivity, efficacy, orresponse of tumor cells such as triple-negative breast tumor cells toanticancer therapy such as triplet therapy.

In one aspect, the present invention provides a method for determiningthe sensitivity of a triple-negative tumor cell to therapy with ananticancer drug, the method comprising:

-   -   (a) lysing the tumor cell to produce a cellular extract;    -   (b) determining the expression level of VEGFR2, c-KIT, HER1,        and/or IGF-1R in the cellular extract; and    -   (c) comparing the expression level of VEGFR2, c-KIT, HER1,        and/or IGF-1R in the cellular extract determined in step (b) to        a reference expression level of VEGFR2, c-KIT, HER1, and/or        IGF-1R,    -   wherein the presence of a low level of VEGFR2 expression, a low        level of c-KIT expression, a high level of HER1 expression,        and/or a low level of IGF-1R expression in the cellular extract        compared to the reference expression level indicates that the        tumor cell is sensitive to the anticancer drug.

In some embodiments, the methods of the present invention may be usefulto aid or assist in determining or predicting the sensitivity of atriple-negative tumor cell to therapy with an anticancer drug. In otherembodiments, the methods of the present invention may be useful forimproving the determination or prediction of the sensitivity of atriple-negative tumor cell to therapy with an anticancer drug.

In another aspect, the present invention provides a method forpredicting the response of a triple-negative breast tumor to therapywith an anticancer drug, the method comprising:

-   -   (a) lysing a tumor cell obtained from the triple-negative breast        tumor to produce a cellular extract;    -   (b) determining the expression level of VEGFR2, c-KIT, HER1,        and/or IGF-1R in the cellular extract; and    -   (c) comparing the expression level of VEGFR2, c-KIT, HER1,        and/or IGF-1R in the cellular extract determined in step (b) to        a reference expression level of VEGFR2, c-KIT, HER1, and/or        IGF-1R,    -   wherein the presence of a low level of VEGFR2 expression, a low        level of c-KIT expression, a high level of HER1 expression,        and/or a low level of IGF-1R expression in the cellular extract        compared to the reference expression level is predictive of        response to therapy with the anticancer drug.

In some embodiments, the methods of the present invention may be usefulto aid or assist in determining or predicting the response of atriple-negative breast tumor to therapy with an anticancer drug. Inother embodiments, the methods of the present invention may be usefulfor improving the determination or prediction of the response of atriple-negative breast tumor to therapy with an anticancer drug.

In a further aspect, the present invention provides a method formonitoring the response to therapy with an anticancer drug in a subjecthaving a triple-negative breast tumor and receiving the anticancer drug,the method comprising:

-   -   (a) lysing a tumor cell obtained from the triple-negative breast        tumor to produce a cellular extract;    -   (b) determining the expression level of VEGFR2, c-KIT, HER1,        and/or IGF-1R in the cellular extract;    -   (c) comparing the expression level of VEGFR2, c-KIT, HER1,        and/or IGF-1R in the cellular extract determined in step (b) to        a reference expression level of VEGFR2, c-KIT, HER1, and/or        IGF-1R or to an expression level of VEGFR2, c-KIT, HER1, and/or        IGF-1R at an earlier time during therapy; and    -   (d) determining whether therapy with the anticancer drug should        be continued or adjusted based upon the comparison in step (c).

In some embodiments, the methods of the present invention may be usefulto aid or assist in monitoring the response of a triple-negative breasttumor to therapy with an anticancer drug. In other embodiments, themethods of the present invention may be useful for improving themonitoring of the response of a triple-negative breast tumor to therapywith an anticancer drug. In certain embodiments, the adjustment oftherapy in step (d) comprises changing a subsequent dose of theanticancer drug or selecting an alternative anticancer drug.

Other objects, features, and advantages of the present invention will beapparent to one of skill in the art from the following detaileddescription and figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the array designs of exemplary slide formats for analyzingtotal and phosphorylated HER1 and HER2 levels.

FIG. 2 shows a schematic of an exemplary proximity assay for detectingphosphorylated HER1. GO, glucose oxidase; HRP, horseradish peroxidase.

FIG. 3 shows a schematic of the Collaborative Enzyme Enhanced ReactiveImmunoAssay (CEER), also known as the COllaborative ProximityImmunoAssay (COPIA). When target proteins are bound to specific captureantibodies printed on nitrocellulose surface after incubating with celllysate, unbound non-target proteins are removed from the slide. One ofthe detector antibodies against alternate epitope on capturedtarget-protein are conjugated with GO. Binding of another detectorantibodies specific to phosphorylated sites on target protein (P) oranother non-overlapping epitope (p) conjugated with HRP completes theformation of immuno-complex necessary for signal generation andsubsequent tyramide mediated signal amplification through GO-HRP enzymechanneling in the presence of glucose. The capture and detectionantibodies were selected to minimize competition between them (i.e., allantibodies can simultaneously bind their corresponding epitope on thesignal transduction protein).

FIG. 4 shows titration curves generated from CEER for ERBB2-T andERBB2-P. These values are used as standards to generate quantitativevalues for clinical samples.

FIG. 5 shows the determination of t-ERBB2 in BT474 cells. The ERBB2-CEERassay was performed using cell lysates prepared from BT474 cells. Thefull-length p185-ERBB2 assay was determined from lysates containing ˜25BT474 cells and the level of t-ERBB2 was determined by analyzing celllysates prepared from ˜250 BT474 cells post immuno-magnetic removal ofp185-ERBB2.

FIG. 6 shows the expression and phosphorylation of t-ERBB2s in patienttumors. ERBB2, t-ERBB2, phosphorylated t-ERBB2. The array configurationis indicated. CEER not only allows differentiation of full-length vs.truncated ERBB2 expression in clinical samples, but also providesvaluable information on the level of phosphorylation in a quantitativemanner.

FIG. 7 shows an exemplary IP-Western for ERBB2 for clinical samples.Anti-ICD-ERBB2 antibodies were used to immuno-precipitate ERBB2receptors and subsequent Western blot analysis was performed usingsecond anti-ICD-ERBB2 antibodies to differentiate full-length t-ERBB2from full-length p185-ERBB2.

FIG. 8 shows that a wide range of pathway protein expression andactivation in 174 BCA samples was observed.

FIG. 9 shows an example of functional pathway profiling by CEER on atriple negative breast cancer core-biopsy sample compared to controlT47D breast cancer cells and human umbilical vein endothelial cells(HUVEC).

FIG. 10 shows the results of a comparison between the Progression FreeSurvival (PFS) for the low and high sample groups for each marker.

FIG. 11 shows the results of another comparison between the PFS for thelow and high sample groups for each marker.

FIG. 12 shows that measuring the expression levels of both c-KIT andVEGFR2 increases the predictive value of determining response to triplettherapy in TNMBC.

FIG. 13 shows that measuring the expression levels of both VEGFR2 andHER1 increases the predictive value of determining response to triplettherapy in TNMBC.

FIG. 14 shows the correlation between increasing levels of (A) totalVEGFR2, (B) total c-KIT, and (C) total HER1 and response to triplettherapy.

The figures and tables from PCT Publication No. WO 2010/132723 areherein incorporated by reference in their entirety for all purposes.

DETAILED DESCRIPTION OF THE INVENTION

I. Introduction

As described above, the activation of signal transduction pathways thatare involved in cell proliferation and the deactivation of pathways thatare involved in cell death are non-limiting examples of molecularfeatures that characterize many different types of cancer. In manycases, the activity of particular signal transduction pathways, andcomponents thereof, may serve as molecular signatures for a given typeof cancer. Such activated components may further provide useful targetsfor therapeutic intervention. Accordingly, knowledge of the activitylevel of a particular signal transduction system within a cancer cellprior to, during, and after treatment provides a physician with highlyrelevant information that may be used to select an appropriate course oftreatment to adopt. Furthermore, the continued monitoring of signaltransduction pathways that are active in cancer cells as treatmentprogresses can provide the physician with additional information on theefficacy of treatment, prompting the physician to either continue aparticular course of treatment or to switch to another line oftreatment, when, for example, cancer cells have become resistant totreatment through further aberrations that activate either the same oranother signal transduction pathway.

Accordingly, the present invention provides methods and compositions fordetecting the expression and/or activation states of one or a pluralityof deregulated signal transduction molecules in tumor tissue orextratumoral cells such as rare circulating cells of a solid tumor in aspecific, multiplex, high-throughput assay. The invention also providesmethods and compositions for the selection of appropriate therapy(single drugs or combinations of drugs) to down-regulate or shut down aderegulated signaling pathway. Thus, the invention may be used tofacilitate the design of personalized therapies for cancer patients.

In certain embodiments, the ability to detect and identify tumor cellsin the circulation through the determination of the activity of signaltransduction pathways at the level of single cells is an importantadvantage of the present invention. Tumor cells are often found in theblood of patients with various early stages of cancer as“micrometastases” (disseminated tumor cells) and are also found inmetastatic cancers. The number of tumor cells in blood will depend onthe stage and type of tumor. While biopsies are typically obtained onprimary tumors, most metastatic tumors are not biopsied, makingmolecular analysis of such tumor samples very difficult. During tumormetastasis, the most aggressive tumor cells leave the primary tumor andtravel through the blood and lymphatic system to reach a distantlocation. Thus, circulating tumor cells from blood represent the mostaggressive and homogenous population of tumor cells. However, the numberof metastatic tumor cells in blood is frequently very low, varying fromone to several thousand cells per milliliter of blood. The ability toisolate and assay signal transduction pathways in such rare cells and toapply this information toward more effective cancer treatments is oneobject of the present invention.

In particular embodiments, the multiplex, high-throughput immunoassaysof the present invention (e.g., Collaborative Enzyme Enhanced ReactiveImmunoAssay (CEER), also known as the COllaborative ProximityImmunoAssay (COPIA)) can detect the level of expression and/oractivation of one or more signal transduction molecules in cellsobtained from tumor tissue (e.g., FNA samples) or in circulating cellsof a solid tumor at the single cell level. In fact, signal transductionmolecules such as EGFR can be detected with a sensitivity of about 100zeptomoles and a linear dynamic range of from about 100 zeptomoles toabout 100 femtomoles. As such, single-cell detection of the expressionand/or activation levels of one or multiple signal transducers in tumorcells facilitates cancer prognosis and diagnosis as well as the designof personalized, targeted therapies.

With regard to breast cancer, current testing options are unsatisfactorybecause treatment of both primary and metastatic tumors in a breastcancer patient is based on a one-time diagnosis from a biopsy sampletaken during an early stage of the disease. In particular, therapeuticintervention for both the early and metastatic stages of breast canceris based solely on the initial diagnosis from the biopsy sample takenduring an early stage of the disease because of the impracticality ofobtaining a biopsy sample from a metastatic cancer patient. However,breast tumors are evolving as a function of time and treatment such thattemporal monitoring of breast tumors is critical for optimal managementof breast cancer patients. For example, a change in the activation stateof one or more of the ErbB (HER) family of receptor tyrosine kinases mayaffect therapy selection at recurrence. Indeed, discordance in HER-2status between primary and metastatic cancer is common because up to 37%of all breast cancer patients change from a HER-2-negative primary tumorto HER-2-positive metastatic cancer. In addition, patients may have denovo resistance or develop acquired resistance to hormonal therapy dueto HER-1 and/or HER-2 activation. In some instances, patients may havede novo resistance or develop acquired resistance to ErbB-targetedtherapies due to the presence of tumor cells expressing p95HER-2. As aresult, there is an unmet clinical need for assays to assist theclinician in prescribing the appropriate cancer therapy at theappropriate time because current technology lacks sensitivity andspecificity, cannot be used to monitor patients on therapy, and do notutilize pathway profiling to guide individualized treatment decisions.

In contrast to currently available breast cancer testing options, themethods of the present invention enable the monitoring of breast cancerpatients through all stages of the disease by providing a “real-timebiopsy” of solid breast tumors using samples such as fine needleaspirates (FNAs) from the tumor and/or circulating tumor cells (CTCs)from blood. As a non-limiting example, the breast cancer assaysdescribed herein can be used in the initial diagnosis of breast cancerin a patient at an early stage of the disease. Selection of a suitablecancer therapy is guided by profiling the expression and/or activationlevels of one or more specific signaling pathways with or withoutanticancer drugs using the single detection and proximity dual detectionassays (e.g., CEER) described herein. Advantageously, the methods of thepresent invention can also be used to monitor the progression and/orregression of the disease because therapeutic intervention may be basedon samples taken at any stage of the disease and analyzed using thesingle detection and proximity dual detection assays (e.g., CEER)described herein. As such, prediction, identification, and/or selectionof suitable cancer therapies for the early and metastatic stages ofbreast cancer is guided by real-time diagnosis and an analysis of theexpression and/or activation status of specific signaling pathwaymolecules.

The methods of the present invention are beneficially tailored toaddress key issues in cancer management and provide a higher standard ofcare for breast cancer patients (e.g., triple-negative metastatic breastcancer (TNMBC) patients) because they: (1) provide increased sensitivity(e.g., single cell detection can be achieved for detecting total and/orphosphorylated signal transduction molecules such as HER1 (EGFR),VEGFR2, and/or c-KIT); (2) provide increased specificity (e.g.,three-antibody proximity assays enhance the specificity for detectingtotal and/or phosphorylated signal transduction molecules); (3) enablepathway profiling (e.g., expression and/or activation status of one ormore specific signal transduction molecules can be detected in FNA orCTCs from patients); and (4) eliminate any issues with obtaining patientsamples (e.g., assays can be performed on a few tumor cells). Althoughany sample may be used in the novel assays described herein, CTCs areparticularly useful because they represent the most aggressive tumorcells, every tumor is known to shed CTCs, they can be the only source ofresidual tumors or hard-to-access metastatic tumors, and they are foundin blood. As such, in certain embodiments, the methods of the presentinvention enable the serial sampling of breast tumor tissues, resultingin valuable information on changes occurring in tumor cells as afunction of time and therapy and providing clinicians with a means tomonitor rapidly evolving cancer pathway signatures.

In sum, the methods of the present invention advantageously provideaccurate selection and monitoring of cancer patients (e.g., TNMBCpatients) most likely to benefit from targeted therapy by performingpathway profiling on tumor cells using multiplexed, antibody-basedsingle detection or proximity assays.

II. Definitions

As used herein, the following terms have the meanings ascribed to themunless specified otherwise.

The term “cancer” is intended to include any member of a class ofdiseases characterized by the uncontrolled growth of aberrant cells. Theterm includes all known cancers and neoplastic conditions, whethercharacterized as malignant, benign, soft tissue, or solid, and cancersof all stages and grades including pre- and post-metastatic cancers.Examples of different types of cancer include, but are not limited to,breast cancer; lung cancer (e.g., non-small cell lung cancer); digestiveand gastrointestinal cancers such as colorectal cancer, gastrointestinalstromal tumors, gastrointestinal carcinoid tumors, colon cancer, rectalcancer, anal cancer, bile duct cancer, small intestine cancer, andstomach (gastric) cancer; esophageal cancer; gallbladder cancer; livercancer; pancreatic cancer; appendix cancer; ovarian cancer; renal cancer(e.g., renal cell carcinoma); cancer of the central nervous system; skincancer; lymphomas; choriocarcinomas; head and neck cancers; osteogenicsarcomas; and blood cancers. As used herein, a “tumor” comprises one ormore cancerous cells. In one preferred embodiment, the breast tumor isderived from a subject with an invasive or in situ form of ductalcarcinoma or lobular carcinoma. In another preferred embodiment, thebreast tumor is derived from a subject with recurrent or metastaticbreast cancer.

The term “analyte” includes any molecule of interest, typically amacromolecule such as a polypeptide, whose presence, amount (expressionlevel), activation state, and/or identity is determined. In certaininstances, the analyte is a signal transduction molecule such as, e.g.,HER1 (EGFR), VEGFR2, or c-KIT.

The term “signal transduction molecule” or “signal transducer” includesproteins and other molecules that carry out the process by which a cellconverts an extracellular signal or stimulus into a response, typicallyinvolving ordered sequences of biochemical reactions inside the cell.Examples of signal transduction molecules include, but are not limitedto, receptor tyrosine kinases such as EGFR (e.g., EGFR/HER1/ErbB1,HER2/Neu/ErbB2, HER3/ErbB3, HER4/ErbB4), VEGFR1/FLT1, VEGFR2/FLK1/KDR,VEGFR3/FLT4, FLT3/FLK2, PDGFR (e.g., PDGFRA, PDGFRB), c-KIT/SCFR, INSR(insulin receptor), IGF-IR, IGF-IIR, IRR (insulin receptor-relatedreceptor), CSF-1R, FGFR 1-4, HGFR 1-2, CCK4, TRK A-C, c-MET, RON, EPHA1-8, EPHB 1-6, AXL, MER, TYRO3, TIE 1-2, TEK, RYK, DDR 1-2, RET, c-ROS,V-cadherin, LTK (leukocyte tyrosine kinase), ALK (anaplastic lymphomakinase), ROR1-2, MUSK, AATYK 1-3, and RTK 106; truncated forms ofreceptor tyrosine kinases such as truncated HER2 receptors with missingamino-terminal extracellular domains (e.g., p95ErbB2 (p95m), p110, p95c,p95n, etc.); receptor tyrosine kinase dimers (e.g., p95HER2/HER3,p95HER2/HER2, HER2/HER2, HER2/HER3, HER1/HER2, HER2/HER3, HER2/HER4,etc.); non-receptor tyrosine kinases such as BCR-ABL, Src, Frk, Btk,Csk, Abl, Zap70, Fes/Fps, Fak, Jak, Ack, and LIMK; tyrosine kinasesignaling cascade components such as AKT (e.g., AKT1, AKT2, AKT3), MEK(MAP2K1), ERK2 (MAPK1), ERK1 (MAPK3), PI3K (e.g., PIK3CA (p110), PIK3R1(p85)), PDK1, PDK2, phosphatase and tensin homolog (PTEN), SGK3, 4E-BP1,P70S6K (e.g., p70 S6 kinase splice variant alpha I), protein tyrosinephosphatases (e.g., PTP1B, PTPN13, BDP1, etc.), RAF, PLA2, MEKK, JNKK,JNK, p38, Shc (p66), Ras (e.g., K-Ras, N-Ras, H-Ras), Rho, Rac1, Cdc42,PLC, PKC, p53, cyclin D1, STAT1, STAT3, phosphatidylinositol4,5-bisphosphate (PIP2), phosphatidylinositol 3,4,5-trisphosphate(PIP3), mTOR, BAD, p21, p2′7, ROCK, IP3, TSP-1, NOS, GSK-3β, RSK 1-3,JNK, c-Jun, Rb, CREB, Ki67, and paxillin; nuclear hormone receptors suchas estrogen receptor (ER), progesterone receptor (PR), androgenreceptor, glucocorticoid receptor, mineralocorticoid receptor, vitamin Areceptor, vitamin D receptor, retinoid receptor, thyroid hormonereceptor, and orphan receptors; nuclear receptor coactivators andrepressors such as amplified in breast cancer-1 (AIB1) and nuclearreceptor corepressor 1 (NCOR), respectively; and combinations thereof.

The term “activation state” refers to whether a particular signaltransduction molecule is activated. Similarly, the term “activationlevel” refers to what extent a particular signal transduction moleculeis activated. The activation state or level typically corresponds to thephosphorylation, ubiquitination, and/or complexation status or level ofone or more (e.g., a plurality of) signal transduction molecules.Non-limiting examples of activation states (listed in parentheses)include: HER1/EGFR (EGFRvIII, phosphorylated (p-) EGFR, EGFR:Shc,ubiquitinated (u-) EGFR, p-EGFRvIII); ErbB2 (p-ErbB2, p95HER2 (truncatedErbB2), p-p95HER2, ErbB2:Shc, ErbB2:PI3K, ErbB2:EGFR, ErbB2:ErbB3,ErbB2:ErbB4); ErbB3 (p-ErbB3, ErbB3:PI3K, p-ErbB3:PI3K, ErbB3:Shc);ErbB4 (p-ErbB4, ErbB4:Shc); c-MET (p-c-MET, c-Met:HGF complex); AKT1(p-AKT1); AKT2 (p-AKT2); AKT3 (p-AKT3); PTEN (p-PTEN); P70S6K(p-P70S6K); MEK (p-MEK); ERK1 (p-ERK1); ERK2 (p-ERK2); PDK1 (p-PDK1);PDK2 (p-PDK2); SGK3 (p-SGK3); 4E-BP1 (p-4E-BP1); PIK3R1 (p-PIK3R1);c-KIT (p-c-KIT); ER (p-ER); IGF-1R (p-IGF-1R, IGF-1R:IRS, IRS:PI3K,p-IRS, IGF-1R:PI3K); INSR (p-INSR); FLT3 (p-FLT3); HGFR1 (p-HGFR1);HGFR2 (p-HGFR2); RET (p-RET); PDGFRA (p-PDGFRA); PDGFRB (p-PDGFRB);VEGFR1 (p-VEGFR1, VEGFR1:PLCγ, VEGFR1:Src); VEGFR2 (p-VEGFR2,VEGFR2:PLCγ, VEGFR2:Src, VEGFR2:heparin sulphate, VEGFR2:VE-cadherin);VEGFR3 (p-VEGFR3); FGFR1 (p-FGFR1); FGFR2 (p-FGFR2); FGFR3 (p-FGFR3);FGFR4 (p-FGFR4); TIE1 (p-TIE1); TIE2 (p-TIE2); EPHA (p-EPHA); EPHB(p-EPHB); GSK-3β(p-GSK-3β); NFKB (p-NFKB), IKB (p-IKB, p-P65:IKB); BAD(p-BAD, BAD:14-3-3); mTOR (p-mTOR); Rsk-1 (p-Rsk-1); Jnk (p-Jnk); P38(p-P38); STAT1 (p-STAT1); STAT3 (p-STAT3); FAK (p-FAK); RB (p-RB); Ki67;p53 (p-p53); CREB (p-CREB); c-Jun (p-c-Jun); c-Src (p-c-Src); paxillin(p-paxillin); GRB2 (p-GRB2), Shc (p-Shc), Ras (p-Ras), GAB1 (p-GAB1),SHP2 (p-SHP2), GRB2 (p-GRB2), CRKL (p-CRKL), PLCγ (p-PLCγ), PKC (e.g.,p-PKCα, p-PKCβ, p-PKCδ), adducin (p-adducin), RB1 (p-RB1), and PYK2(p-PYK2).

As used herein, the term “dilution series” is intended to include aseries of descending concentrations of a particular sample (e.g., celllysate) or reagent (e.g., antibody). A dilution series is typicallyproduced by a process of mixing a measured amount of a startingconcentration of a sample or reagent with a diluent (e.g., dilutionbuffer) to create a lower concentration of the sample or reagent, andrepeating the process enough times to obtain the desired number ofserial dilutions. The sample or reagent can be serially diluted at least2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 500, or1000-fold to produce a dilution series comprising at least 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40,45, or 50 descending concentrations of the sample or reagent. Forexample, a dilution series comprising a 2-fold serial dilution of acapture antibody reagent at a 1 mg/ml starting concentration can beproduced by mixing an amount of the starting concentration of captureantibody with an equal amount of a dilution buffer to create a 0.5 mg/mlconcentration of the capture antibody, and repeating the process toobtain capture antibody concentrations of 0.25 mg/ml, 0.125 mg/ml,0.0625 mg/ml, 0.0325 mg/ml, etc.

The term “superior dynamic range” as used herein refers to the abilityof an assay to detect a specific analyte in as few as one cell or in asmany as thousands of cells. For example, the immunoassays describedherein possess superior dynamic range because they advantageously detecta particular signal transduction molecule of interest in about 1-10,000cells (e.g., about 1, 5, 10, 25, 50, 75, 100, 250, 500, 750, 1000, 2500,5000, 7500, or 10,000 cells) using a dilution series of capture antibodyconcentrations.

The term “sample” as used herein includes any biological specimenobtained from a patient. Samples include, without limitation, wholeblood, plasma, serum, red blood cells, white blood cells (e.g.,peripheral blood mononuclear cells), ductal lavage fluid, nippleaspirate, lymph (e.g., disseminated tumor cells of the lymph node), bonemarrow aspirate, saliva, urine, stool (i.e., feces), sputum, bronchiallavage fluid, tears, fine needle aspirate (e.g., harvested by randomperiareolar fine needle aspiration), any other bodily fluid, a tissuesample (e.g., tumor tissue) such as a biopsy of a tumor (e.g., needlebiopsy) or a lymph node (e.g., sentinel lymph node biopsy), a tissuesample (e.g., tumor tissue) such as a surgical resection of a tumor, andcellular extracts thereof. In some embodiments, the sample is wholeblood or a fractional component thereof such as plasma, serum, or a cellpellet. In certain instances, the sample is obtained by isolatingcirculating cells of a solid tumor from whole blood or a cellularfraction thereof using any technique known in the art. In otherembodiments, the sample is a formalin fixed paraffin embedded (FFPE)tumor tissue sample, e.g., from a solid tumor of the breast.

A “biopsy” refers to the process of removing a tissue sample fordiagnostic or prognostic evaluation, and to the tissue specimen itself.Any biopsy technique known in the art can be applied to the methods andcompositions of the present invention. The biopsy technique applied willgenerally depend on the tissue type to be evaluated and the size andtype of the tumor (i.e., solid or suspended (i.e., blood or ascites)),among other factors.

Representative biopsy techniques include excisional biopsy, incisionalbiopsy, needle biopsy (e.g., core needle biopsy, fine-needle aspirationbiopsy, etc.), surgical biopsy, and bone marrow biopsy. Biopsytechniques are discussed, for example, in Harrison's Principles ofInternal Medicine, Kasper, et al., eds., 16th ed., 2005, Chapter 70, andthroughout Part V. One skilled in the art will appreciate that biopsytechniques can be performed to identify cancerous and/or precancerouscells in a given tissue sample.

As used herein, the term “circulating cells” comprises extratumoralcells that have either metastasized or micrometastasized from a solidtumor. Examples of circulating cells include, but are not limited to,circulating tumor cells, cancer stem cells, and/or cells that aremigrating to the tumor (e.g., circulating endothelial progenitor cells,circulating endothelial cells, circulating pro-angiogenic myeloid cells,circulating dendritic cells, etc.). Patient samples containingcirculating cells can be obtained from any accessible biological fluid(e.g., whole blood, serum, plasma, sputum, bronchial lavage fluid,urine, nipple aspirate, lymph, saliva, fine needle aspirate, etc.). Incertain instances, the whole blood sample is separated into a plasma orserum fraction and a cellular fraction (i.e., cell pellet). The cellularfraction typically contains red blood cells, white blood cells, and/orcirculating cells of a solid tumor such as circulating tumor cells(CTCs), circulating endothelial cells (CECs), circulating endothelialprogenitor cells (CEPCs), cancer stem cells (CSCs), disseminated tumorcells of the lymph node, and combinations thereof. The plasma or serumfraction usually contains, inter alia, nucleic acids (e.g., DNA, RNA)and proteins that are released by circulating cells of a solid tumor.

Circulating cells are typically isolated from a patient sample using oneor more separation methods including, for example, immunomagneticseparation (see, e.g., Racila et al., Proc. Natl. Acad. Sci. USA,95:4589-4594 (1998); Bilkenroth et al., Int. J. Cancer, 92:577-582(2001)), the CellTracks® System by Immunicon (Huntingdon Valley, Pa.),microfluidic separation (see, e.g., Mohamed et al., IEEE Trans.Nanobiosci., 3:251-256 (2004); Lin et al., Abstract No. 5147, 97th AACRAnnual Meeting, Washington, D.C. (2006)), FACS (see, e.g., Mancuso etal., Blood, 97:3658-3661 (2001)), density gradient centrifugation (see,e.g., Baker et al., Clin. Cancer Res., 13:4865-4871 (2003)), anddepletion methods (see, e.g., Meye et al., Int. J. Oncol., 21:521-530(2002)).

Signal transduction molecules of interest are typically extractedshortly after the circulating cells are isolated to preserve their insitu activation state, preferably within about 24, 6, or 1 hr, and morepreferably within about 30, 15, or 5 minutes. The isolated cells mayalso be incubated with one or more growth factors, usually at nanomolarto micromolar concentrations, for about 1-30 minutes to resuscitate orstimulate activation of the signal transduction molecules (see, e.g.,Irish et al., Cell, 118:217-228 (2004)). For example, to evaluatepotential anticancer therapies for an individual patient, the isolatedcells can be incubated with one or more anticancer drugs at varyingdoses. Growth factor stimulation can then be performed for a few minutes(e.g., about 1-5 minutes) or for several hours (e.g., about 1-6 hours).The differential activation of signaling pathways with and withoutanticancer drugs can aid in the selection of a suitable cancer therapyat the proper dose for each individual patent. Circulating cells canalso be isolated from a patient sample during anticancer drug treatmentand stimulated with one or more growth factors to determine whether achange in therapy should be implemented.

The term “subject” or “patient” or “individual” typically includeshumans, but can also include other animals such as, e.g., otherprimates, rodents, canines, felines, equines, ovines, porcines, and thelike.

An “array” or “microarray” comprises a distinct set and/or dilutionseries of capture antibodies immobilized or restrained on a solidsupport such as, for example, glass (e.g., a glass slide), plastic,chips, pins, filters, beads (e.g., magnetic beads, polystyrene beads,etc.), paper, membrane (e.g., nylon, nitrocellulose, polyvinylidenefluoride (PVDF), etc.), fiber bundles, or any other suitable substrate.The capture antibodies are generally immobilized or restrained on thesolid support via covalent or noncovalent interactions (e.g., ionicbonds, hydrophobic interactions, hydrogen bonds, Van der Waals forces,dipole-dipole bonds). In certain instances, the capture antibodiescomprise capture tags which interact with capture agents bound to thesolid support. The arrays used in the assays described herein typicallycomprise a plurality of different capture antibodies and/or captureantibody concentrations that are coupled to the surface of a solidsupport in different known/addressable locations.

The term “capture antibody” is intended to include an immobilizedantibody which is specific for (i.e., binds, is bound by, or forms acomplex with) one or more analytes of interest in a sample such as acellular extract. In particular embodiments, the capture antibody isrestrained on a solid support in an array. Suitable capture antibodiesfor immobilizing any of a variety of signal transduction molecules on asolid support are available from Upstate (Temecula, Calif.), Biosource(Camarillo, Calif.), Cell Signaling Technologies (Danvers, Mass.), R&DSystems (Minneapolis, Minn.), Lab Vision (Fremont, Calif.), Santa CruzBiotechnology (Santa Cruz, Calif.), Sigma (St. Louis, Mo.), and BDBiosciences (San Jose, Calif.).

The term “detection antibody” as used herein includes an antibodycomprising a detectable label which is specific for (i.e., binds, isbound by, or forms a complex with) one or more analytes of interest in asample. The term also encompasses an antibody which is specific for oneor more analytes of interest, wherein the antibody can be bound byanother species that comprises a detectable label. Examples ofdetectable labels include, but are not limited to, biotin/streptavidinlabels, nucleic acid (e.g., oligonucleotide) labels, chemically reactivelabels, fluorescent labels, enzyme labels, radioactive labels, andcombinations thereof. Suitable detection antibodies for detecting theactivation state and/or total amount of any of a variety of signaltransduction molecules are available from Upstate (Temecula, Calif.),Biosource (Camarillo, Calif.), Cell Signaling Technologies (Danvers,Mass.), R&D Systems (Minneapolis, Minn.), Lab Vision (Fremont, Calif.),Santa Cruz Biotechnology (Santa Cruz, Calif.), Sigma (St. Louis, Mo.),and BD Biosciences (San Jose, Calif.). As a non-limiting example,phospho-specific antibodies against various phosphorylated forms ofsignal transduction molecules such as EGFR, c-KIT, c-Src, FLK-1, PDGFRA,PDGFRB, AKT, MAPK, PTEN, Raf, and MEK are available from Santa CruzBiotechnology.

The term “activation state-dependent antibody” includes a detectionantibody which is specific for (i.e., binds, is bound by, or forms acomplex with) a particular activation state of one or more analytes ofinterest in a sample. In preferred embodiments, the activationstate-dependent antibody detects the phosphorylation, ubiquitination,and/or complexation state of one or more analytes such as one or moresignal transduction molecules. In some embodiments, the phosphorylationof members of the EGFR family of receptor tyrosine kinases and/or theformation of heterodimeric complexes between EGFR family members isdetected using activation state-dependent antibodies. In particularembodiments, activation state-dependent antibodies are useful fordetecting one or more sites of phosphorylation in one or more of thefollowing signal transduction molecules (phosphorylation sitescorrespond to the position of the amino acid in the human proteinsequence): EGFR/HER1/ErbB 1 (e.g., tyrosine (Y) 1068); ErbB2/HER2 (e.g.,Y1248); ErbB3/HER3 (e.g., Y1289); ErbB4/HER4 (e.g., Y1284); c-Met (e.g.,Y1003, Y1230, Y1234, Y1235, and/or Y1349); SGK3 (e.g., threonine (T) 256and/or serine (S) 422); 4E-BP1 (e.g., T70); ERK1 (e.g., T185, Y187,T202, and/or Y204); ERK2 (e.g., T185, Y187, T202, and/or Y204); MEK(e.g., S217 and/or S221); PIK3R1 (e.g., Y688); PDK1 (e.g., S241); P70S6K(e.g., T229, T389, and/or S421); PTEN (e.g., S380); AKT1 (e.g., S473and/or T308); AKT2 (e.g., S474 and/or T309); AKT3 (e.g., S472 and/orT305); GSK-3β (e.g., S9); NFKB (e.g., S536); IKB (e.g., S32); BAD (e.g.,S112 and/or S136); mTOR (e.g., S2448); Rsk-1 (e.g., T357 and/or S363);Jnk (e.g., T183 and/or Y185); P38 (e.g., T180 and/or Y182); STAT3 (e.g.,Y705 and/or S727); FAK (e.g., Y397, Y576, S722, Y861, and/or S910); RB(e.g., S249, T252, 5612, and/or S780); RB1 (e.g., S780); adducin (e.g.,S662 and/or S724); PYK2 (e.g., Y402 and/or Y881); PKCα (e.g., S657);PKCα/β (e.g., T368 and/or T641); PKCβ (e.g., T505); p53 (e.g., S392and/or S20); CREB (e.g., S133); c-Jun (e.g., S63); c-Src (e.g., Y416);and paxillin (e.g., Y31 and/or Y118).

The term “activation state-independent antibody” includes a detectionantibody which is specific for (i.e., binds, is bound by, or forms acomplex with) one or more analytes of interest in a sample irrespectiveof their activation state. For example, the activation state-independentantibody can detect both phosphorylated and unphosphorylated forms ofone or more analytes such as one or more signal transduction molecules.

The term “nucleic acid” or “polynucleotide” includesdeoxyribonucleotides or ribonucleotides and polymers thereof in eithersingle- or double-stranded form such as, for example, DNA and RNA.Nucleic acids include nucleic acids containing known nucleotide analogsor modified backbone residues or linkages, which are synthetic,naturally occurring, and non-naturally occurring, and which have similarbinding properties as the reference nucleic acid. Examples of suchanalogs include, without limitation, phosphorothioates,phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,2′-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). Unlessspecifically limited, the term encompasses nucleic acids containingknown analogues of natural nucleotides that have similar bindingproperties as the reference nucleic acid. Unless otherwise indicated, aparticular nucleic acid sequence also implicitly encompassesconservatively modified variants thereof and complementary sequences aswell as the sequence explicitly indicated.

The term “oligonucleotide” includes a single-stranded oligomer orpolymer of RNA, DNA, RNA/DNA hybrid, and/or a mimetic thereof. Incertain instances, oligonucleotides are composed of naturally-occurring(i.e., unmodified) nucleobases, sugars, and internucleoside (backbone)linkages. In certain other instances, oligonucleotides comprise modifiednucleobases, sugars, and/or internucleoside linkages.

As used herein, the term “mismatch motif” or “mismatch region” refers toa portion of an oligonucleotide that does not have 100% complementarityto its complementary sequence. An oligonucleotide may have at least one,two, three, four, five, six, or more mismatch regions. The mismatchregions may be contiguous or may be separated by 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, or more nucleotides. The mismatch motifs or regions maycomprise a single nucleotide or may comprise two, three, four, five, ormore nucleotides.

The phrase “stringent hybridization conditions” refers to conditionsunder which an oligonucleotide will hybridize to its complementarysequence, but to no other sequences. Stringent conditions aresequence-dependent and will be different in different circumstances.Longer sequences hybridize specifically at higher temperatures. Anextensive guide to the hybridization of nucleic acids is found inTijssen, Techniques in Biochemistry and Molecular Biology—Hybridizationwith Nucleic Probes, “Overview of principles of hybridization and thestrategy of nucleic acid assays” (1993). Generally, stringent conditionsare selected to be about 5-10° C. lower than the thermal melting point(T_(m)) for the specific sequence at a defined ionic strength pH. TheT_(m) is the temperature (under defined ionic strength, pH, and nucleicconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at T_(m), 50% of the probes are occupied atequilibrium). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. For selective orspecific hybridization, a positive signal is at least two timesbackground, preferably 10 times background hybridization.

The terms “substantially identical” or “substantial identity,” in thecontext of two or more nucleic acids, refer to two or more sequences orsubsequences that are the same or have a specified percentage ofnucleotides that are the same (i.e., at least about 60%, preferably atleast about 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity over aspecified region) when compared and aligned for maximum correspondenceover a comparison window or designated region as measured using asequence comparison algorithm or by manual alignment and visualinspection. This definition, when the context indicates, also refersanalogously to the complement of a sequence. Preferably, the substantialidentity exists over a region that is at least about 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 75, or 100 nucleotides in length.

The term “incubating” is used synonymously with “contacting” and“exposing” and does not imply any specific time or temperaturerequirements unless otherwise indicated.

“Receptor tyrosine kinases” or “RTKs” include a family of fifty-six (56)proteins characterized by a transmembrane domain and a tyrosine kinasemotif. RTKs function in cell signaling and transmit signals regulatinggrowth, differentiation, adhesion, migration, and apoptosis. Themutational activation and/or overexpression of receptor tyrosine kinasestransforms cells and often plays a crucial role in the development ofcancers. RTKs have become targets of various molecularly targeted agentssuch as trastuzumab, cetuximab, gefitinib, erlotinib, sunitinib,imatinib, nilotinib, and the like. One well-characterized signaltransduction pathway is the MAP kinase pathway, which is responsible fortransducing the signal from epidermal growth factor (EGF) to thepromotion of cell proliferation in cells.

The term “progression free survival” or “PFS” includes the length oftime during and after a treatment of a disease (e.g., cancer) in which apatient is living with the disease without additional symptoms of thedisease.

The term “triple-negative” in the context of the present inventionincludes a tumor cell (e.g., a circulating tumor cell), a tumor, or acancer such as triple-negative metastatic breast cancer (TNMBC) in whichthere is no detectable expression of estrogen receptor (ER),progesterone receptor (PR), or human epidermal growth factor receptor 2(HER2).

III. Description of the Embodiments

The present invention provides methods for detecting the status (e.g.,expression and/or activation levels) of components of signaltransduction pathways in tumor cells derived from tumor tissue orcirculating cells of a solid tumor with an assay such as a specific,multiplex, high-throughput proximity assay as described herein. Thepresent invention also provides methods for selecting appropriatetherapies to downregulate one or more deregulated signal transductionpathways. Thus, certain embodiments of the invention may be used tofacilitate the design of personalized therapies based on the particularmolecular signature provided by the collection of total and/or activatedsignal transduction proteins in a given patient's tumor (e.g., atriple-negative breast tumor).

In particular aspects, the present invention provides molecular markers(biomarkers) that enable the determination or prediction of whether aparticular cancer can respond or is likely to respond favorably to oneor more anticancer drugs such as, e.g., a combination of bevacizumab(Avastin®), carboplatin, and paclitaxel (e.g., Abraxane® or nabP)(“triplet therapy”). In specific embodiments, measuring the level ofexpression and/or activation of at least one, two, or more (e.g., all)of VEGFR2, c-KIT, HER1, and/or IGF-1R is particularly useful forselecting a suitable anticancer drug and/or identifying or predictingefficacy or a response thereto in cells such as triple-negative tumorcells.

In one aspect, the present invention provides a method for determiningthe sensitivity of a triple-negative tumor cell to therapy with ananticancer drug, the method comprising:

-   -   (a) lysing the tumor cell to produce a cellular extract;    -   (b) determining the expression level of one or more analytes        selected from the group consisting of VEGFR2, c-KIT, HER1,        and/or IGF-1R in the cellular extract; and    -   (c) comparing the expression level of VEGFR2, c-KIT, HER1,        and/or IGF-1R in the cellular extract determined in step (b) to        a reference expression level of VEGFR2, c-KIT, HER1, and/or        IGF-1R,    -   wherein the presence of a low level of VEGFR2 expression, a low        level of c-KIT expression, a high level of HER1 expression,        and/or a low level of IGF-1R expression in the cellular extract        compared to the reference expression level indicates that the        tumor cell is sensitive to the anticancer drug.

In some embodiments, the presence of a medium to high level of VEGFR2expression, a medium to high level of c-KIT expression, a low to mediumlevel of HER1 expression, and/or a medium to high level of IGF-1Rexpression in the cellular extract compared to the reference expressionlevel indicates that the tumor cell is resistant to the anticancer drug.In one particular embodiment, the method comprises determining theexpression level of a combination of analytes comprising, consistingessentially of, or consisting of VEGFR2 and c-KIT in the cellularextract. In another particular embodiment, the method comprisesdetermining the expression level of a combination of analytescomprising, consisting essentially of, or consisting of VEGFR2 and HER1in the cellular extract. In yet another particular embodiment, themethod comprises determining the expression level of a combination ofanalytes comprising, consisting essentially of, or consisting of VEGFR2,c-KIT, and HER1 in the cellular extract. In a further particularembodiment, the method comprises determining the expression level of acombination of analytes comprising, consisting essentially of, orconsisting of VEGFR2, c-KIT, HER1, and IGF-1R in the cellular extract.In certain instances, the method of the present invention furthercomprises determining the activation level of at least one, two, or more(e.g., all) of VEGFR2, c-KIT, HER1, IGF-1R, and/or AKT in the cellularextract. In other instances, the method further comprises contacting thetumor cell with the anticancer drug prior to step (a).

In other embodiments, the tumor cell is a breast cancer cell. In certaininstances, the tumor cell is a fine needle aspirate (FNA) cell obtainedfrom a tumor such as a triple-negative breast tumor or a circulatingtumor cell (CTC) obtained from a bodily fluid sample. The tumor cell istypically isolated from a sample including whole blood, serum, plasma,or tumor tissue. In particular embodiments, the sample is obtained froma subject with triple-negative metastatic breast cancer (TNMBC).

In another aspect, the present invention provides a method forpredicting the response of a triple-negative breast tumor to therapywith an anticancer drug, the method comprising:

-   -   (a) lysing a tumor cell obtained from the triple-negative breast        tumor to produce a cellular extract;    -   (b) determining the expression level of VEGFR2, c-KIT, HER1,        and/or IGF-1R in the cellular extract; and    -   (c) comparing the expression level of VEGFR2, c-KIT, HER1,        and/or IGF-1R in the cellular extract determined in step (b) to        a reference expression level of VEGFR2, c-KIT, HER1, and/or        IGF-1R,    -   wherein the presence of a low level of VEGFR2 expression, a low        level of c-KIT expression, a high level of HER1 expression,        and/or a low level of IGF-1R expression in the cellular extract        compared to the reference expression level is predictive of        response to therapy with the anticancer drug.

In some embodiments, the presence of a medium to high level of VEGFR2expression, a medium to high level of c-KIT expression, a low to mediumlevel of HER1 expression, and/or a medium to high level of IGF-1Rexpression in the cellular extract is predictive of a lack of responseto therapy with the anticancer drug. In one particular embodiment, themethod comprises determining the expression level of a combination ofanalytes comprising, consisting essentially of, or consisting of VEGFR2and c-KIT in the cellular extract. In another particular embodiment, themethod comprises determining the expression level of a combination ofanalytes comprising, consisting essentially of, or consisting of VEGFR2and HER1 in the cellular extract. In yet another particular embodiment,the method comprises determining the expression level of a combinationof analytes comprising, consisting essentially of, or consisting ofVEGFR2, c-KIT, and HER1 in the cellular extract. In a further particularembodiment, the method comprises determining the expression level of acombination of analytes comprising, consisting essentially of, orconsisting of VEGFR2, c-KIT, HER1, and IGF-1R in the cellular extract.In certain instances, the method of the present invention furthercomprises determining the activation level of at least one, two, or more(e.g., all) of VEGFR2, c-KIT, HER1, IGF-1R, and/or AKT in the cellularextract. In other instances, the method further comprises incubating thetumor cell obtained from the triple-negative breast tumor with theanticancer drug prior to step (a).

In other embodiments, the tumor cell is a fine needle aspirate (FNA)cell obtained from a tumor such as a triple-negative breast tumor or acirculating tumor cell (CTC) obtained from a bodily fluid sample. Thetumor cell is typically isolated from a sample including whole blood,serum, plasma, or tumor tissue. In particular embodiments, the sample isobtained from a subject with triple-negative metastatic breast cancer(TNMBC).

In some instances, the presence of a low level of VEGFR2 expression ispredictive of a longer duration of progression free survival (PFS). Inother instances, the presence of a low level of c-KIT expression ispredictive of a longer duration of PFS. In further instances, thepresence of a high level of HER1 expression is predictive of a longerduration of PFS. In yet other instances, the presence of a low level ofIGF-1R expression is predictive of a longer duration of PFS. Inparticular instances, the presence of a low level of VEGFR2 expressionin combination with the presence of a low level of c-KIT expressionand/or a high level of HER1 expression is predictive of a longerduration of PFS compared to the expression level of VEGFR2, c-KIT, orHER1 alone.

In certain embodiments, the methods of the present invention (e.g.,methods for determining the sensitivity of a triple-negative tumor cellto therapy with an anticancer drug and for predicting the response of atriple-negative breast tumor to therapy with an anticancer drug) mayfurther comprise step (d) of providing the result of the comparisonobtained in step (c) to a user (e.g., a clinician such as an oncologistor a general practitioner) in a readable format. In certain embodiments,the methods of the present invention may further comprise sending orreporting the result of the comparison obtained in step (c) to aclinician, e.g., an oncologist or a general practitioner. In otherinstances, the methods of the present invention may further compriserecording or storing the result of the comparison obtained in step (c)in a computer database or other suitable machine or device for storinginformation, e.g., at a laboratory.

In particular embodiments, the expression level of VEGFR2, c-KIT, HER1,and/or IGF-1R is determined by detecting total protein levels of VEGFR2,c-KIT, HER1, and/or IGF-1R, e.g., using an immunoassay withanalyte-specific antibodies. Total expression level and/or status can bedetermined using any of a variety of techniques. As non-limitingexamples, the expression level of VEGFR2, c-KIT, HER1, and/or IGF-1R canbe determined with a single detection assay or with a proximity dualdetection assay as described herein. In preferred embodiments, theproximity dual detection assay is a Collaborative Enzyme EnhancedReactive ImmunoAssay (CEER).

In some embodiments, the expression (e.g., total) level and/oractivation (e.g., phosphorylation) level of the one or more analytes isexpressed as a relative fluorescence unit (RFU) value that correspondsto the signal intensity for a particular analyte of interest that isdetermined using, e.g., CEER. In other embodiments, the expression leveland/or activation level of the one or more analytes is quantitated bycalibrating or normalizing the RFU value that is determined using, e.g.,a proximity assay such as CEER, against a standard curve generated forthe particular analyte of interest. In certain instances, the RFU valuecan be calculated based upon a standard curve.

In further embodiments, the expression level and/or activation level ofthe one or more analytes is expressed as “low”, “medium”, or “high” thatcorresponds to increasing signal intensity for a particular analyte ofinterest that is determined using, e.g., a proximity assay such as CEER.In some instances, an undetectable or minimally detectable level ofexpression or activation of a particular analyte of interest that isdetermined using, e.g., a proximity assay such as CEER, may be expressedas “undetectable”. In other instances, a low level of expression oractivation of a particular analyte of interest that is determined using,e.g., a proximity assay such as CEER, may be expressed as “low”. In yetother instances, a moderate level of expression or activation of aparticular analyte of interest that is determined using, e.g., aproximity assay such as CEER, may be expressed as “medium”. In still yetother instances, a moderate to high level of expression or activation ofa particular analyte of interest that is determined using, e.g., aproximity assay such as CEER, may be expressed as “medium to high”. Infurther instances, a very high level of expression or activation of aparticular analyte of interest that is determined using, e.g., aproximity assay such as CEER, may be expressed as “high”.

In particular embodiments, the reference expression level and/oractivation level of a particular analyte of interest is calculated fromone or more standard curves generated from a sample such as, forexample, a cancer cell line. As a non-limiting example, for each assayused to determine the expression level or activation level of aparticular analyte of interest, a sigmoidal standard curve can begenerated from one or multiple (e.g., two, three, four, five, six,seven, etc.) concentrations of serially diluted cell lysates preparedfrom a cancer cell line. In preferred embodiments, the cancer cell lineexpresses one or more analytes of interest, e.g., VEGFR2, c-KIT, HER1,and/or IGF-1R. Each curve can be plotted as a function of signalintensity vs. log concentration derived units, and CU (Computed Unit)can be calculated based on the standard curve. Example 7 provides a moredetailed description of the quantitation of the expression and/oractivation levels of a particular analyte of interest against a standardcurve generated for the particular analyte of interest.

In certain embodiments, the expression level or activation level of aparticular analyte of interest, when expressed as “low”, “medium”, or“high”, may correspond to a level of expression or activation that is atleast about 0; 5,000; 10,000; 15,000; 20;000; 25,000; 30,000; 35,000;40,000; 45,000; 50,000; 60,000; 70;000; 80,000; 90,000; 100,000 RFU; ormore, e.g., when compared to a reference expression level and/oractivation level for that particular analyte of interest in a negativecontrol (e.g., an IgG control), in a standard curve generated for theanalyte of interest (e.g., a standard curve generated from a cancer cellline), in a positive control such as a pan-CK control, in the presenceof an anticancer drug, and/or in the absence of an anticancer drug. Insome instances, the correlation is analyte-specific. As a non-limitingexample, a “low” level of expression or activation determined using,e.g., a proximity assay such as CEER, may correspond 10,000 RFUs inexpression or activation for one analyte and 50,000 RFUs for anotheranalyte when compared to a reference expression or activation level.

In certain embodiments, the expression or activation level of aparticular analyte of interest may correspond to a level of expressionor activation referred to as “low”, “medium” or “high” that is relativeto a reference expression level or activation level for that particularanalyte of interest, e.g., when compared to a negative control such asan IgG control, when compared to a standard curve generated for theanalyte of interest (e.g., a standard curve generated from a cancer cellline), when compared to a positive control such as a pan-CK control,when compared to an expression or activation level determined in thepresence of an anticancer drug, and/or when compared to an expression oractivation level determined in the absence of an anticancer drug. Insome instances, the correlation is analyte-specific. As a non-limitingexample, a “low” level of expression or activation determined using,e.g., a proximity assay such as CEER, may correspond to a 2-foldincrease in expression or activation for one analyte and a 5-foldincrease for another analyte when compared to a reference expression oractivation level.

In certain embodiments, the expression or activation level of aparticular analyte of interest may correspond to a level of expressionor activation that is compared to a reference expression level and/oractivation level for that particular analyte of interest in a negativecontrol (e.g., an IgG control), in a standard curve generated for theanalyte of interest (e.g., a standard curve generated from a cancer cellline), in a positive control such as a pan-CK control, in the presenceof an anticancer drug, and/or in the absence of an anticancer drug.

In certain embodiments, a higher level of expression or activation of aparticular analyte of interest is considered to be present in a sample(e.g., a cellular extract) when the expression or activation level is atleast about 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5,9, 9.5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or 100-fold higher (e.g.,about 1.5-3,2-3, 2-4, 2-5, 2-10, 2-20, 2-50, 3-5,3-10, 3-20, 3-50, 4-5,4-10, 4-20, 4-50, 5-10, 5-15, 5-20, or 5-50-fold higher) than thereference expression or activation level for that particular analyte ofinterest in a negative control (e.g., an IgG control), in a standardcurve generated for the analyte of interest (e.g., a standard curvegenerated from a cancer cell line), in a positive control (e.g., apan-CK control), in the presence of an anticancer drug, and/or in theabsence of an anticancer drug.

In other embodiments, a lower level of expression or activation of aparticular analyte of interest is considered to be present in a sample(e.g., a cellular extract) when the expression or activation level is atleast about 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5,9, 9.5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or 100-fold lower (e.g.,about 1.5-3,2-3, 2-4, 2-5, 2-10, 2-20, 2-50, 3-5, 3-10, 3-20, 3-50, 4-5,4-10, 4-20, 4-50, 5-10, 5-15, 5-20, or 5-50-fold lower) than thereference expression or activation level for that particular analyte ofinterest in a negative control (e.g., an IgG control), in a standardcurve generated for the analyte of interest (e.g., a standard curvegenerated from a cancer cell line), in a positive control (e.g., apan-CK control), in the presence of an anticancer drug, and/or in theabsence of an anticancer drug.

In further embodiments, the reference expression or activation level ofa particular analyte of interest is a cutoff value. In some instances,the cutoff value includes a number chosen on the basis of populationanalysis of a particular analyte of interest that is used for comparisonto the expression or activation level of that analyte in the cellularextract. As a non-limiting example, a cutoff value can be derived bydividing the expression or activation level of a particular analyte ofinterest from a population of individuals into “high” and “low” groupsand selected to be at or close to the median expression or activationlevel of that analyte in the population. The expression or activationlevel of the analyte of interest in the cellular extract can be comparedto the cutoff value and determined to be a “high” and “low” level ofexpression or activation based on whether the expression or activationlevel of the analyte in the cellular extract is above (e.g., “high”) orbelow (e.g., “low”) the cutoff value. Example 5 provides one exemplaryembodiment of calculating, selecting, and using cutoff values inaccordance with the methods of the present invention. In otherembodiments, the cutoff value can be derived from a standard curvegenerated for a particular analyte of interest (e.g., a standard curvegenerated from a cancer cell line) and compared to the expression oractivation level of that analyte in the cellular extract. Those of skillin the art will recognize that a cutoff value can be determinedaccording to the needs of the user and characteristics of the analyzedpopulation.

In some embodiments, the anticancer drug comprises one or more agentsthat interfere with the function of abnormally expressed and/oractivated signal transduction pathway components in cancer cells.Non-limiting examples of such agents include those listed below in Table1 of PCT Publication No. WO 2010/132723, the disclosure of which isherein incorporated by reference in its entirety for all purposes.

In certain embodiments, the anticancer drug comprises an anti-signalingagent (i.e., a cytostatic drug) such as a monoclonal antibody or atyrosine kinase inhibitor; an anti-proliferative agent; achemotherapeutic agent (i.e., a cytotoxic drug); a hormonal therapeuticagent; a radiotherapeutic agent; a vaccine; and/or any other compoundwith the ability to reduce or abrogate the uncontrolled growth ofaberrant cells such as cancerous cells. In some embodiments, theisolated cells are treated with one or more anti-signaling agents,anti-proliferative agents, and/or hormonal therapeutic agents incombination with at least one chemotherapeutic agent.

Examples of anti-signaling agents suitable for use in the presentinvention include, without limitation, monoclonal antibodies such astrastuzumab (Herceptin®), pertuzumab (2C4), alemtuzumab (Campath®),bevacizumab (Avastin®), cetuximab (Erbitux®), gemtuzumab (Mylotarg®),panitumumab (Vectibix™), rituximab (Rituxan®), and tositumomab(BEXXAR®); tyrosine kinase inhibitors such as gefitinib (Iressa®),sunitinib (Sutent®), erlotinib (Tarceva®), lapatinib (GW-572016;Tykerb®), canertinib (CI 1033), semaxinib (SU5416), vatalanib(PTK787/ZK222584), sorafenib (BAY 43-9006; Nexavar®), imatinib mesylate(Gleevec®), leflunomide (SU101), vandetanib (ZACTIMA™; ZD6474),pelitinib, CP-654577, CP-724714, HKI-272, PKI-166, AEE788, BMS-599626,HKI-357, BIBW 2992, ARRY-334543, JNJ-26483327, and JNJ-26483327; andcombinations thereof.

Exemplary anti-proliferative agents include mTOR inhibitors such assirolimus (rapamycin), temsirolimus (CCI-779), everolimus (RAD001),BEZ235, and XL765; AKT inhibitors such as1L6-hydroxymethyl-chiro-inositol-2-(R)-2-O-methyl-3-O-octadecyl-sn-glycerocarbonate,9-methoxy-2-methylellipticinium acetate,1,3-dihydro-1-(1-((4-(6-phenyl-1H-imidazo[4,5-g]quinoxalin-7-yl)phenyl)methyl)-4-piperidinyl)-2H-benzimidazol-2-one,10-(4′-(N-diethylamino)butyl)-2-chlorophenoxazine, 3-formylchromonethiosemicarbazone (Cu(II)Cl₂ complex), API-2, a 15-mer peptide derivedfrom amino acids 10-24 of the proto-oncogene TCL1 (Hiromura et al., J.Biol. Chem., 279:53407-53418 (2004), KP372-1, and the compoundsdescribed in Kozikowski et al., J. Am. Chem. Soc., 125:1144-1145 (2003)and Kau et al., Cancer Cell, 4:463-476 (2003); PI3K inhibitors such asPX-866, wortmannin, LY 294002, quercetin, tetrodotoxin citrate,thioperamide maleate, GDC-0941 (957054-30-7), IC87114, PI-103, PIK93,BEZ235 (NVP-BEZ235), TGX-115, ZSTK474, (−)-deguelin, NU 7026, myricetin,tandutinib, GDC-0941 bismesylate, GSK690693, KU-55933, MK-2206,OSU-03012, perifosine, triciribine, XL-147, PIK75, TGX-221, NU 7441, PI828, XL-765, and WHI-P 154; MEK inhibitors such as PD98059, ARRY-162,RDEA119, U0126, GDC-0973, PD184161, AZD6244, AZD8330, PD0325901, andARRY-142886; and combinations thereof.

Non-limiting examples of pan-HER inhibitors include PF-00299804,neratinib (HKI-272), AC480 (BMS-599626), BMS-690154, PF-02341066,HM781-36B, CI-1033, BIBW-2992, and combinations thereof.

Non-limiting examples of chemotherapeutic agents include platinum-baseddrugs (e.g., oxaliplatin, cisplatin, carboplatin, spiroplatin,iproplatin, satraplatin, etc.), alkylating agents (e.g.,cyclophosphamide, ifosfamide, chlorambucil, busulfan, melphalan,mechlorethamine, uramustine, thiotepa, nitrosoureas, etc.),anti-metabolites (e.g., 5-fluorouracil, azathioprine, 6-mercaptopurine,methotrexate, leucovorin, capecitabine, cytarabine, floxuridine,fludarabine, gemcitabine (Gemzar®), pemetrexed (ALIMTA®), raltitrexed,etc.), plant alkaloids (e.g., vincristine, vinblastine, vinorelbine,vindesine, podophyllotoxin, paclitaxel (Taxol®), docetaxel (Taxotere®),etc.), topoisomerase inhibitors (e.g., irinotecan, topotecan, amsacrine,etoposide (VP16), etoposide phosphate, teniposide, etc.), antitumorantibiotics (e.g., doxorubicin, adriamycin, daunorubicin, epirubicin,actinomycin, bleomycin, mitomycin, mitoxantrone, plicamycin, etc.),pharmaceutically acceptable salts thereof, stereoisomers thereof,derivatives thereof, analogs thereof, and combinations thereof.

Examples of hormonal therapeutic agents include, without limitation,aromatase inhibitors (e.g., aminoglutethimide, anastrozole (Arimidex®),letrozole (Femara®), vorozole, exemestane (Aromasin®),4-androstene-3,6,17-trione (6-OXO), 1,4,6-androstatrien-3,17-dione(ATD), formestane (Lentaron®), etc.), selective estrogen receptormodulators (e.g., bazedoxifene, clomifene, fulvestrant, lasofoxifene,raloxifene, tamoxifen, toremifene, etc.), steroids (e.g.,dexamethasone), finasteride, and gonadotropin-releasing hormone agonists(GnRH) such as goserelin, pharmaceutically acceptable salts thereof,stereoisomers thereof, derivatives thereof, analogs thereof, andcombinations thereof.

Non-limiting examples of cancer vaccines useful in the present inventioninclude ANYARA from Active Biotech, DCVax-LB from NorthwestBiotherapeutics, EP-2101 from IDM Pharma, GV1001 from Pharmexa, IO-2055from Idera Pharmaceuticals, INGN 225 from Introgen Therapeutics andStimuvax from Biomira/Merck.

Examples of radiotherapeutic agents include, but are not limited to,radionuclides such as ⁴⁷Sc, ⁶⁴Cu, ⁶⁷Cu, ⁸⁹Sr, ⁸⁶Y, ⁸⁷Y, ⁹⁰Y, ¹⁰⁵Rh,¹¹¹Ag, ¹¹¹In, ^(117m)Sn, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re,²¹¹At, and ²¹²Bi, optionally conjugated to antibodies directed againsttumor antigens.

In preferred embodiments, the anticancer drug is a combination ofbevacizumab (Avastin®), carboplatin, and paclitaxel (“triplet therapy”).In some instances, the paclitaxel is a nanoparticle albumin-bound (nab)paclitaxel (Abraxane® or nabP). In other embodiments, the anticancerdrug comprises one or more of the following: bevacizumab (Avastin®),carboplatin, paclitaxel (e.g., nabP), iniparib (BSI 201;4-iodo-3-nitrobenzamide), NK012 (an SN-38-releasing nanodeviceconstructed by covalently attaching SN-38 to the block copolymerPEG-PGlu, followed by self-assembly of amphiphilic block copolymers inaqueous media), glembatumumab vedotin, (also known as CDX-011 orCR011-vcMMAE; human monoclonal antibody glembatumumab (CR011) linked tomonomethyl auristatin E (MMAE) that targets cancer cells expressingtransmembrane glycoprotein NMB), or combinations thereof. In oneparticular embodiment, the anticancer drug is a combination of iniparib(a PARP inhibitor), gemcitabine (Gemzar®), and carboplatin.

In some embodiments, the methods further comprise determining theexpression and/or activation level of one or more additional signaltransduction molecules in the cellular extract. Non-limiting examples ofadditional signal transduction molecules that can be interrogated forexpression (e.g., total amount) levels and/or activation (e.g.,phosphorylation) levels in a sample such as a cellular extract includereceptor tyrosine kinases, non-receptor tyrosine kinases, tyrosinekinase signaling cascade components, nuclear hormone receptors, nuclearreceptor coactivators, nuclear receptor repressors, and combinationsthereof. Specific examples of signal transduction molecules and pathwaysthat may be interrogated using the present invention include those shownin Table 2 of PCT Publication No. WO 2010/132723, the disclosure ofwhich is herein incorporated by reference in its entirety for allpurposes. In particular embodiments, the one or more additional signaltransduction molecules is selected from the group consisting of HER2,p95HER2, HER3, HER4, PI3K, AKT, MEK, PTEN, SGK3, 4E-BP1, ERK2 (MAPK1),ERK1 (MAPK3), PDK1, P70S6K, GSK-3β, Shc, c-MET, VEGFR1, VEGFR3, areceptor dimer, and combinations thereof.

In certain embodiments, the present invention further comprisesdetermining the expression (e.g., total) level and/or activation (e.g.,phosphorylation) level of one or more (e.g., at least about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more) additional analytesin the cellular extract. In some embodiments, the one or more (e.g., atleast about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, ormore) additional analytes comprises one or more signal transductionmolecules selected from the group consisting of receptor tyrosinekinases, non-receptor tyrosine kinases, tyrosine kinase signalingcascade components, nuclear hormone receptors, nuclear receptorcoactivators, nuclear receptor repressors, and combinations thereof.

In particular embodiments, the present invention further comprisesdetermining the expression (e.g., total) level and/or activation (e.g.,phosphorylation) level of one or any combination of 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 35, 40, 45, 50, or more of the following additionalanalytes in a cellular extract: HER2, p95HER2, HER3, HER4, PI3K, AKT,MEK, PTEN, SGK3, 4E-BP1, ERK2 (MAPK1), ERK1 (MAPK3), PDK1, P70S6K,GSK-3β, Shc, c-MET, VEGFR1, VEGFR3, PDK2, Raf, SRC, NFkB-IkB, mTOR,EPH-A, EPH-B, EPH-C, EPH-D, FLT-3, TIE-1, TIE-2, c-FMS, Abl, FTL 3, RET,FGFR1, FGFR2, FGFR3, FGFR4, ER, PR, NCOR, AIB1, RON, PIP2, PIP3, p27,protein tyrosine phosphatases (e.g., PTP1B, PTPN13, BDP1, etc.),receptor dimers, and combinations thereof.

IV. Construction of Antibody Arrays

In certain aspects, the expression level and/or activation state of oneor more (e.g., a plurality) of analytes (e.g., signal transductionmolecules) in a cellular extract of tumor cells such as breast cancercells is detected using an antibody-based array comprising a dilutionseries of capture antibodies restrained on a solid support. The arraystypically comprise a plurality of different capture antibodies at arange of capture antibody concentrations that are coupled to the surfaceof the solid support in different addressable locations. In oneembodiment, the array comprises capture antibodies for detecting and/orquantifying the expression and/or activation of at least one or more ofVEGFR2, c-KIT, HER1, and/or IGF-1R and one or more controls such as,e.g., a negative control (e.g., an IgG control), a standard curvegenerated for the analyte of interest, and/or a positive control (e.g.,a pan-CK control).

In one particular embodiment, the present invention provides anaddressable array having superior dynamic range comprising a pluralityof dilution series of capture antibodies restrained on a solid support,in which the capture antibodies in each dilution series are specific forone or more analytes corresponding to a component of a signaltransduction pathway and other target proteins. In various aspects, thisembodiment includes arrays that comprise components of signaltransduction pathways characteristic of particular tumors, e.g., signaltransduction pathways active in breast cancer cells. Thus, the presentinvention may be advantageously practiced wherein each signaltransduction molecule or other protein of interest with a potentialexpression or activation defect causing breast cancer is represented ona single array or chip. In some aspects, the components of a givensignal transduction pathway active in a particular tumor cell arearrayed in a linear sequence that corresponds to the sequence in whichinformation is relayed through a signal transduction pathway within acell. Examples of such arrays are described herein and also shown inFIGS. 5-9 of PCT Publication No. WO2009/108637, the disclosure of whichis herein incorporated by reference in its entirety for all purposes.The capture antibodies specific for one or more components of a givensignal transduction pathway active in a particular tumor cell can alsobe printed in a randomized fashion to minimize any surface-relatedartifacts.

The solid support can comprise any suitable substrate for immobilizingproteins. Examples of solid supports include, but are not limited to,glass (e.g., a glass slide), plastic, chips, pins, filters, beads,paper, membranes, fiber bundles, gels, metal, ceramics, and the like.Membranes such nylon (Biotrans™, ICN Biomedicals, Inc. (Costa Mesa,Calif.); Zeta-Probe®, Bio-Rad Laboratories (Hercules, Calif.)),nitrocellulose (Protran®, Whatman Inc. (Florham Park, N.J.)), and PVDF(Immobilon™, Millipore Corp. (Billerica, Mass.)) are suitable for use assolid supports in the arrays of the present invention. Preferably, thecapture antibodies are restrained on glass slides coated with anitrocellulose polymer, e.g., FAST® Slides, which are commerciallyavailable from Whatman Inc. (Florham Park, N.J.).

Particular aspects of the solid support which are desirable include theability to bind large amounts of capture antibodies and the ability tobind capture antibodies with minimal denaturation. Another suitableaspect is that the solid support displays minimal “wicking” whenantibody solutions containing capture antibodies are applied to thesupport. A solid support with minimal wicking allows small aliquots ofcapture antibody solution applied to the support to result in small,defined spots of immobilized capture antibody.

The capture antibodies are typically directly or indirectly (e.g., viacapture tags) restrained on the solid support via covalent ornoncovalent interactions (e.g., ionic bonds, hydrophobic interactions,hydrogen bonds, Van der Waals forces, dipole-dipole bonds). In someembodiments, the capture antibodies are covalently attached to the solidsupport using a homobifunctional or heterobifunctional crosslinker usingstandard crosslinking methods and conditions. Suitable crosslinkers arecommercially available from vendors such as, e.g., Pierce Biotechnology(Rockford, Ill.).

Methods for generating arrays suitable for use in the present inventioninclude, but are not limited to, any technique used to construct proteinor nucleic acid arrays. In some embodiments, the capture antibodies arespotted onto an array using a microspotter, which are typically roboticprinters equipped with split pins, blunt pins, or ink jet printing.Suitable robotic systems for printing the antibody arrays describedherein include the PixSys 5000 robot (Cartesian Technologies; Irvine,Calif.) with ChipMaker2 split pins (TeleChem International; Sunnyvale,Calif.) as well as other robotic printers available from BioRobics(Woburn, Mass.) and Packard Instrument Co. (Meriden, Conn.). Preferably,at least 2, 3, 4, 5, or 6 replicates of each capture antibody dilutionare spotted onto the array.

Another method for generating arrays suitable for use in the presentinvention comprises dispensing a known volume of a capture antibodydilution at each selected array position by contacting a capillarydispenser onto a solid support under conditions effective to draw adefined volume of liquid onto the support, wherein this process isrepeated using selected capture antibody dilutions at each selectedarray position to create a complete array. The method may be practicedin forming a plurality of such arrays, where the solution-depositingstep is applied to a selected position on each of a plurality of solidsupports at each repeat cycle. A further description of such a methodcan be found, e.g., in U.S. Pat. No. 5,807,522.

In certain instances, devices for printing on paper can be used togenerate the antibody arrays. For example, the desired capture antibodydilution can be loaded into the printhead of a desktop jet printer andprinted onto a suitable solid support (see, e.g., Silzel et al., Clin.Chem., 44:2036-2043 (1998)).

In some embodiments, the array generated on the solid support has adensity of at least about 5 spots/cm², and preferably at least about 10,20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170,180, 190, 200, 210, 220, 230, 250, 275, 300, 325, 350, 375, 400, 425,450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 2000,3000, 4000, 5000, 6000, 7000, 8000 or 9000, or 10,000 spots/cm².

In certain instances, the spots on the solid support each represents adifferent capture antibody. In certain other instances, multiple spotson the solid support represent the same capture antibody, e.g., as adilution series comprising a series of descending capture antibodyconcentrations.

Additional examples of methods for preparing and constructing antibodyarrays on solid supports are described in U.S. Pat. Nos. 6,197,599,6,777,239, 6,780,582, 6,897,073, 7,179,638, and 7,192,720; U.S. PatentPublication Nos. 20060115810, 20060263837, 20060292680, and 20070054326;and Varnum et al., Methods Mol. Biol., 264:161-172 (2004).

Methods for scanning antibody arrays are known in the art and include,without limitation, any technique used to scan protein or nucleic acidarrays. Microarray scanners suitable for use in the present inventionare available from PerkinElmer (Boston, MA), Agilent Technologies (PaloAlto, CA), Applied Precision (Issaquah, WA), GSI Lumonics Inc.(Billerica, MA), and Axon Instruments (Union City, CA). As anon-limiting example, a GSI ScanArray™ 3000 for fluorescence detectioncan be used with ImaGene™ software for quantitation.

V. Single Detection Assays

In some embodiments, the assay for detecting the expression and/oractivation level of one or more analytes (e.g., one or more signaltransduction molecules) of interest in a cellular extract of cells suchas tumor cells is a multiplex, high-throughput two-antibody assay havingsuperior dynamic range. As a non-limiting example, the two antibodiesused in the assay can comprise: (1) a capture antibody specific for aparticular analyte of interest; and (2) a detection antibody specificfor an activated form of the analyte (i.e., activation state-dependentantibody). The activation state-dependent antibody is capable ofdetecting, for example, the phosphorylation, ubiquitination, and/orcomplexation state of the analyte. Alternatively, the detection antibodycomprises an activation state-independent antibody, which detects thetotal amount of the analyte in the cellular extract. The activationstate-independent antibody is generally capable of detecting both theactivated and non-activated forms of the analyte.

In one particular embodiment, the two-antibody assay for detecting theexpression or activation level of an analyte of interest comprises:

-   -   (i) incubating the cellular extract with one or a plurality of        dilution series of capture antibodies to form a plurality of        captured analytes;    -   (ii) incubating the plurality of captured analytes with        detection antibodies specific for the corresponding analytes to        form a plurality of detectable captured analytes, wherein the        detection antibodies comprise activation state-dependent        antibodies for detecting the activation (e.g., phosphorylation)        level of the analyte or activation state-independent antibodies        for detecting the expression level (e.g., total amount) of the        analyte;    -   (iii) incubating the plurality of detectable captured analytes        with first and second members of a signal amplification pair to        generate an amplified signal; and    -   (iv) detecting the amplified signal generated from the first and        second members of the signal amplification pair.

The two-antibody assays described herein are typically antibody-basedarrays which comprise a plurality of different capture antibodies at arange of capture antibody concentrations that are coupled to the surfaceof a solid support in different addressable locations. Examples ofsuitable solid supports for use in the present invention are describedabove.

The capture antibodies and detection antibodies are preferably selectedto minimize competition between them with respect to analyte binding(i.e., both capture and detection antibodies can simultaneously bindtheir corresponding signal transduction molecules).

In one embodiment, the detection antibodies comprise a first member of abinding pair (e.g., biotin) and the first member of the signalamplification pair comprises a second member of the binding pair (e.g.,streptavidin). The binding pair members can be coupled directly orindirectly to the detection antibodies or to the first member of thesignal amplification pair using methods well-known in the art. Incertain instances, the first member of the signal amplification pair isa peroxidase (e.g., horseradish peroxidase (HRP), catalase,chloroperoxidase, cytochrome c peroxidase, eosinophil peroxidase,glutathione peroxidase, lactoperoxidase, myeloperoxidase, thyroidperoxidase, deiodinase, etc.), and the second member of the signalamplification pair is a tyramide reagent (e.g., biotin-tyramide). Inthese instances, the amplified signal is generated by peroxidaseoxidization of the tyramide reagent to produce an activated tyramide inthe presence of hydrogen peroxide (H₂O₂).

The activated tyramide is either directly detected or detected upon theaddition of a signal-detecting reagent such as, for example, astreptavidin-labeled fluorophore or a combination of astreptavidin-labeled peroxidase and a chromogenic reagent. Examples offluorophores suitable for use in the present invention include, but arenot limited to, an Alexa Fluor® dye (e.g., Alexa Fluor® 555),fluorescein, fluorescein isothiocyanate (FITC), Oregon Green™;rhodamine, Texas red, tetrarhodamine isothiocynate (TRITC), a CyDye™fluor (e.g., Cy2, Cy3, Cy5), and the like. The streptavidin label can becoupled directly or indirectly to the fluorophore or peroxidase usingmethods well-known in the art. Non-limiting examples of chromogenicreagents suitable for use in the present invention include3,3′,5,5′-tetramethylbenzidine (TMB), 3,3′-diaminobenzidine (DAB),2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS),4-chloro-1-napthol (4CN), and/or porphyrinogen.

An exemplary protocol for performing the two-antibody assays describedherein is provided in Example 3 of PCT Publication No. WO2009/108637,the disclosure of which is herein incorporated by reference in itsentirety for all purposes.

In another embodiment of a two-antibody approach, the present inventionprovides a method for detecting the expression or activation level of atruncated receptor, the method comprising:

-   -   (i) incubating the cellular extract with a plurality of beads        specific for an extracellular domain (ECD) binding region of a        full-length receptor;    -   (ii) removing the plurality of beads from the cellular extract,        thereby removing the full-length receptor to form a cellular        extract devoid of the full-length receptor;    -   (iii) incubating the cellular extract devoid of the full-length        receptor with a dilution series of one or a plurality of capture        antibodies specific for an intracellular domain (ICD) binding        region of the full-length receptor to form a plurality of        captured truncated receptors;    -   (iv) incubating the plurality of captured truncated receptors        with detection antibodies specific for an ICD binding region of        the full-length receptor to form a plurality of detectable        captured truncated receptors, wherein the detection antibodies        comprise activation state-dependent antibodies for detecting the        activation (e.g., phosphorylation) level of the truncated        receptor or activation state-independent antibodies for        detecting the expression level (e.g., total amount) of the        truncated receptor;    -   (v) incubating the plurality of detectable captured truncated        receptors with first and second members of a signal        amplification pair to generate an amplified signal; and    -   (vi) detecting an amplified signal generated from the first and        second members of the signal amplification pair.

In certain embodiments, the truncated receptor is p95HER2 and thefull-length receptor is HER2. In certain other embodiments, theplurality of beads specific for an extracellular domain (ECD) bindingregion comprises a streptavidin-biotin pair, wherein the biotin isattached to the bead and the biotin is attached to an antibody (e.g.,wherein the antibody is specific for the ECD binding region of thefull-length receptor).

FIG. 14A of PCT Publication No. WO2009/108637, the disclosure of whichis herein incorporated by reference in its entirety for all purposes,shows that beads coated with an antibody directed to the extracellulardomain (ECD) of a receptor of interest binds the full-length receptor(e.g., HER2), but not the truncated receptor (e.g., p95HER2) to removeany full-length receptor from the assay. FIG. 14B of PCT Publication No.WO2009/108637 shows that the truncated receptor (e.g., p95HER2), oncebound to a capture antibody, may then be detected by a detectionantibody that is specific for the intracellular domain (ICD) of thefull-length receptor (e.g., HER2). The detection antibody may bedirectly conjugated to horseradish peroxidase (HRP). Tyramide signalamplification (TSA) may then be performed to generate a signal to bedetected. The expression level or activation state of the truncatedreceptor (e.g., p95HER2) can be interrogated to determine, e.g., itstotal concentration or its phosphorylation state, ubiquitination state,and/or complexation state.

In another embodiment, the present invention provides kits forperforming the two-antibody assays described above comprising: (a) adilution series of one or a plurality of capture antibodies restrainedon a solid support; and (b) one or a plurality of detection antibodies(e.g., activation state-independent antibodies and/or activationstate-dependent antibodies). In some instances, the kits can furthercontain instructions for methods of using the kit to detect theexpression levels and/or activation states of one or a plurality ofsignal transduction molecules of cells such as tumor cells. The kits mayalso contain any of the additional reagents described above with respectto performing the specific methods of the present invention such as, forexample, first and second members of the signal amplification pair,tyramide signal amplification reagents, wash buffers, etc.

VI. Proximity Dual Detection Assays

In some embodiments, the assay for detecting the expression and/oractivation level of one or more analytes (e.g., one or more signaltransduction molecules) of interest in a cellular extract of cells suchas tumor cells is a multiplex, high-throughput proximity (i.e.,three-antibody) assay having superior dynamic range. As a non-limitingexample, the three antibodies used in the proximity assay can comprise:(1) a capture antibody specific for a particular analyte of interest;(2) a detection antibody specific for an activated form of the analyte(i.e., activation state-dependent antibody); and (3) a detectionantibody which detects the total amount of the analyte (i.e., activationstate-independent antibody). The activation state-dependent antibody iscapable of detecting, e.g., the phosphorylation, ubiquitination, and/orcomplexation state of the analyte, while the activationstate-independent antibody is capable of detecting the total amount(i.e., both the activated and non-activated forms) of the analyte.

In one particular embodiment, the proximity assay for detecting theactivation level or status of an analyte of interest comprises:

-   -   (i) incubating the cellular extract with one or a plurality of        dilution series of capture antibodies to form a plurality of        captured analytes;    -   (ii) incubating the plurality of captured analytes with        detection antibodies comprising one or a plurality of activation        state-independent antibodies and one or a plurality of        activation state-dependent antibodies specific for the        corresponding analytes to form a plurality of detectable        captured analytes,    -   wherein the activation state-independent antibodies are labeled        with a facilitating moiety, the activation state-dependent        antibodies are labeled with a first member of a signal        amplification pair, and the facilitating moiety generates an        oxidizing agent which channels to and reacts with the first        member of the signal amplification pair;    -   (iii) incubating the plurality of detectable captured analytes        with a second member of the signal amplification pair to        generate an amplified signal; and    -   (iv) detecting the amplified signal generated from the first and        second members of the signal amplification pair.

In another particular embodiment, the proximity assay for detecting theactivation level or status of an analyte of interest that is a truncatedreceptor comprises:

-   -   (i) incubating the cellular extract with a plurality of beads        specific for an extracellular domain (ECD) binding region of a        full-length receptor;    -   (ii) removing the plurality of beads from the cellular extract,        thereby removing the full-length receptor to form a cellular        extract devoid of the full-length receptor;    -   (iii) incubating the cellular extract devoid of the full-length        receptor with one or a plurality of capture antibodies specific        for an intracellular domain (ICD) binding region of the        full-length receptor to form a plurality of captured truncated        receptors;    -   (iv) incubating the plurality of captured truncated receptors        with detection antibodies comprising one or a plurality of        activation state-independent antibodies and one or a plurality        of activation state-dependent antibodies specific for an ICD        binding region of the full-length receptor to form a plurality        of detectable captured truncated receptors,    -   wherein the activation state-independent antibodies are labeled        with a facilitating moiety, the activation state-dependent        antibodies are labeled with a first member of a signal        amplification pair, and the facilitating moiety generates an        oxidizing agent which channels to and reacts with the first        member of the signal amplification pair;    -   (v) incubating the plurality of detectable captured truncated        receptors with a second member of the signal amplification pair        to generate an amplified signal; and    -   (vi) detecting the amplified signal generated from the first and        second members of the signal amplification pair.

In certain embodiments, the truncated receptor is p95HER2 and thefull-length receptor is HER2. In certain other embodiments, theplurality of beads specific for an extracellular domain (ECD) bindingregion comprises a streptavidin-biotin pair, wherein the biotin isattached to the bead and the biotin is attached to an antibody (e.g.,wherein the antibody is specific for the ECD binding region of thefull-length receptor).

In alternative embodiments, the activation state-dependent antibodiescan be labeled with a facilitating moiety and the activationstate-independent antibodies can be labeled with a first member of asignal amplification pair.

As another non-limiting example, the three antibodies used in theproximity assay can comprise: (1) a capture antibody specific for aparticular analyte of interest; (2) a first detection antibody whichdetects the total amount of the analyte (i.e., a first activationstate-independent antibody); and (3) a second detection antibody whichdetects the total amount of the analyte (i.e., a second activationstate-independent antibody). In preferred embodiments, the first andsecond activation state-independent antibodies recognize different(e.g., distinct) epitopes on the analyte.

In one particular embodiment, the proximity assay for detecting theexpression level of an analyte of interest comprises:

-   -   (i) incubating the cellular extract with one or a plurality of        dilution series of capture antibodies to form a plurality of        captured analytes;    -   (ii) incubating the plurality of captured analytes with        detection antibodies comprising one or a plurality of first and        second activation state-independent antibodies specific for the        corresponding analytes to form a plurality of detectable        captured analytes,    -   wherein the first activation state-independent antibodies are        labeled with a facilitating moiety, the second activation        state-independent antibodies are labeled with a first member of        a signal amplification pair, and the facilitating moiety        generates an oxidizing agent which channels to and reacts with        the first member of the signal amplification pair;    -   (iii) incubating the plurality of detectable captured analytes        with a second member of the signal amplification pair to        generate an amplified signal; and    -   (iv) detecting the amplified signal generated from the first and        second members of the signal amplification pair.

In another particular embodiment, the proximity assay for detecting theexpression level of an analyte of interest that is a truncated receptorcomprises:

-   -   (i) incubating the cellular extract with a plurality of beads        specific for an extracellular domain (ECD) binding region of a        full-length receptor;    -   (ii) removing the plurality of beads from the cellular extract,        thereby removing the full-length receptor to form a cellular        extract devoid of the full-length receptor;    -   (iii) incubating the cellular extract devoid of the full-length        receptor with one or a plurality of capture antibodies specific        for an intracellular domain (ICD) binding region of the        full-length receptor to form a plurality of captured truncated        receptors;    -   (iv) incubating the plurality of captured truncated receptors        with detection antibodies comprising one or a plurality of first        and second activation state-independent antibodies specific for        an ICD binding region of the full-length receptor to form a        plurality of detectable captured truncated receptors,    -   wherein the first activation state-independent antibodies are        labeled with a facilitating moiety, the second activation        state-independent antibodies are labeled with a first member of        a signal amplification pair, and the facilitating moiety        generates an oxidizing agent which channels to and reacts with        the first member of the signal amplification pair;    -   (v) incubating the plurality of detectable captured truncated        receptors with a second member of the signal amplification pair        to generate an amplified signal; and    -   (vi) detecting the amplified signal generated from the first and        second members of the signal amplification pair.

In certain embodiments, the truncated receptor is p95HER2 and thefull-length receptor is HER2. In certain other embodiments, theplurality of beads specific for an extracellular domain (ECD) bindingregion comprises a streptavidin-biotin pair, wherein the biotin isattached to the bead and the biotin is attached to an antibody (e.g.,wherein the antibody is specific for the ECD binding region of thefull-length receptor).

In alternative embodiments, the first activation state-independentantibodies can be labeled with a first member of a signal amplificationpair and the second activation state-independent antibodies can belabeled with a facilitating moiety.

The proximity assays described herein are typically antibody-basedarrays which comprise one or a plurality of different capture antibodiesat a range of capture antibody concentrations that are coupled to thesurface of a solid support in different addressable locations. Examplesof suitable solid supports for use in the present invention aredescribed above.

The capture antibodies, activation state-independent antibodies, andactivation state-dependent antibodies are preferably selected tominimize competition between them with respect to analyte binding (i.e.,all antibodies can simultaneously bind their corresponding signaltransduction molecules).

In some embodiments, activation state-independent antibodies fordetecting activation levels of one or more of the analytes or,alternatively, first activation state-independent antibodies fordetecting expression levels of one or more of the analytes furthercomprise a detectable moiety. In such instances, the amount of thedetectable moiety is correlative to the amount of one or more of theanalytes in the cellular extract. Examples of detectable moietiesinclude, but are not limited to, fluorescent labels, chemically reactivelabels, enzyme labels, radioactive labels, and the like. Preferably, thedetectable moiety is a fluorophore such as an Alexa Fluor® dye (e.g.,Alexa Fluor® 647), fluorescein, fluorescein isothiocyanate (FITC),Oregon Green™; rhodamine, Texas red, tetrarhodamine isothiocynate(TRITC), a CyDye™ fluor (e.g., Cy2, Cy3, Cy5), and the like. Thedetectable moiety can be coupled directly or indirectly to theactivation state-independent antibodies using methods well-known in theart.

In certain instances, activation state-independent antibodies fordetecting activation levels of one or more of the analytes or,alternatively, first activation state-independent antibodies fordetecting expression levels of one or more of the analytes are directlylabeled with the facilitating moiety. The facilitating moiety can becoupled to activation state-independent antibodies using methodswell-known in the art. A suitable facilitating moiety for use in thepresent invention includes any molecule capable of generating anoxidizing agent which channels to (i.e., is directed to) and reacts with(i.e., binds, is bound by, or forms a complex with) another molecule inproximity (i.e., spatially near or close) to the facilitating moiety.Examples of facilitating moieties include, without limitation, enzymessuch as glucose oxidase or any other enzyme that catalyzes anoxidation/reduction reaction involving molecular oxygen (O₂) as theelectron acceptor, and photosensitizers such as methylene blue, rosebengal, porphyrins, squarate dyes, phthalocyanines, and the like.Non-limiting examples of oxidizing agents include hydrogen peroxide(H₂O₂), a singlet oxygen, and any other compound that transfers oxygenatoms or gains electrons in an oxidation/reduction reaction. Preferably,in the presence of a suitable substrate (e.g., glucose, light, etc.),the facilitating moiety (e.g., glucose oxidase, photosensitizer, etc.)generates an oxidizing agent (e.g., hydrogen peroxide (H₂O₂), singleoxygen, etc.) which channels to and reacts with the first member of thesignal amplification pair (e.g., horseradish peroxidase (HRP), haptenprotected by a protecting group, an enzyme inactivated by thioetherlinkage to an enzyme inhibitor, etc.) when the two moieties are inproximity to each other.

In certain other instances, activation state-independent antibodies fordetecting activation levels of one or more of the analytes or,alternatively, first activation state-independent antibodies fordetecting expression levels of one or more of the analytes areindirectly labeled with the facilitating moiety via hybridizationbetween an oligonucleotide linker conjugated to the activationstate-independent antibodies and a complementary oligonucleotide linkerconjugated to the facilitating moiety. The oligonucleotide linkers canbe coupled to the facilitating moiety or to the activationstate-independent antibodies using methods well-known in the art. Insome embodiments, the oligonucleotide linker conjugated to thefacilitating moiety has 100% complementarity to the oligonucleotidelinker conjugated to the activation state-independent antibodies. Inother embodiments, the oligonucleotide linker pair comprises at leastone, two, three, four, five, six, or more mismatch regions, e.g., uponhybridization under stringent hybridization conditions. One skilled inthe art will appreciate that activation state-independent antibodiesspecific for different analytes can either be conjugated to the sameoligonucleotide linker or to different oligonucleotide linkers.

The length of the oligonucleotide linkers that are conjugated to thefacilitating moiety or to the activation state-independent antibodiescan vary. In general, the linker sequence can be at least about 5, 10,15, 20, 25, 30, 35, 40, 45, 50, 75, or 100 nucleotides in length.Typically, random nucleic acid sequences are generated for coupling. Asa non-limiting example, a library of oligonucleotide linkers can bedesigned to have three distinct contiguous domains: a spacer domain;signature domain; and conjugation domain. Preferably, theoligonucleotide linkers are designed for efficient coupling withoutdestroying the function of the facilitating moiety or activationstate-independent antibodies to which they are conjugated.

The oligonucleotide linker sequences can be designed to prevent orminimize any secondary structure formation under a variety of assayconditions. Melting temperatures are typically carefully monitored foreach segment within the linker to allow their participation in theoverall assay procedures. Generally, the range of melting temperaturesof the segment of the linker sequence is between 1-10° C. Computeralgorithms (e.g., OLIGO 6.0) for determining the melting temperature,secondary structure, and hairpin structure under defined ionicconcentrations can be used to analyze each of the three differentdomains within each linker. The overall combined sequences can also beanalyzed for their structural characterization and their comparabilityto other conjugated oligonucleotide linker sequences, e.g., whether theywill hybridize under stringent hybridization conditions to acomplementary oligonucleotide linker.

The spacer region of the oligonucleotide linker provides adequateseparation of the conjugation domain from the oligonucleotidecrosslinking site. The conjugation domain functions to link moleculeslabeled with a complementary oligonucleotide linker sequence to theconjugation domain via nucleic acid hybridization. The nucleicacid-mediated hybridization can be performed either before or afterantibody-analyte (i.e., antigen) complex formation, providing a moreflexible assay format. Unlike many direct antibody conjugation methods,linking relatively small oligonucleotides to antibodies or othermolecules has minimal impact on the specific affinity of antibodiestowards their target analyte or on the function of the conjugatedmolecules.

In some embodiments, the signature sequence domain of theoligonucleotide linker can be used in complex multiplexed proteinassays. Multiple antibodies can be conjugated with oligonucleotidelinkers with different signature sequences. In multiplex immunoassays,reporter oligonucleotide sequences labeled with appropriate probes canbe used to detect cross-reactivity between antibodies and their antigensin the multiplex assay format.

Oligonucleotide linkers can be conjugated to antibodies or othermolecules using several different methods. For example, oligonucleotidelinkers can be synthesized with a thiol group on either the 5′ or 3′end. The thiol group can be deprotected using reducing agents (e.g.,TCEP-HCl) and the resulting linkers can be purified by using a desaltingspin column. The resulting deprotected oligonucleotide linkers can beconjugated to the primary amines of antibodies or other types ofproteins using heterobifunctional cross linkers such as SMCC.Alternatively, 5′-phosphate groups on oligonucleotides can be treatedwith water-soluble carbodiimide EDC to form phosphate esters andsubsequently coupled to amine-containing molecules. In certaininstances, the diol on the 3′-ribose residue can be oxidized to aldehydegroups and then conjugated to the amine groups of antibodies or othertypes of proteins using reductive amination. In certain other instances,the oligonucleotide linker can be synthesized with a biotin modificationon either the 3′ or 5′ end and conjugated to streptavidin-labeledmolecules.

Oligonucleotide linkers can be synthesized using any of a variety oftechniques known in the art, such as those described in Usman et al., J.Am. Chem. Soc., 109:7845 (1987); Scaringe et al., Nucl. Acids Res.,18:5433 (1990); Wincott et al., Nucl. Acids Res., 23:2677-2684 (1995);and Wincott et al., Methods Mol. Bio., 74:59 (1997). In general, thesynthesis of oligonucleotides makes use of common nucleic acidprotecting and coupling groups, such as dimethoxytrityl at the 5′-endand phosphoramidites at the 3′-end. Suitable reagents foroligonucleotide synthesis, methods for nucleic acid deprotection, andmethods for nucleic acid purification are known to those of skill in theart.

In certain instances, activation state-dependent antibodies fordetecting activation levels of one or more of the analytes or,alternatively, second activation state-independent antibodies fordetecting expression levels of one or more of the analytes are directlylabeled with the first member of the signal amplification pair. Thesignal amplification pair member can be coupled to activationstate-dependent antibodies to detect activation levels or secondactivation state-independent antibodies to detect expression levelsusing methods well-known in the art. In certain other instances,activation state-dependent antibodies or second activationstate-independent antibodies are indirectly labeled with the firstmember of the signal amplification pair via binding between a firstmember of a binding pair conjugated to the activation state-dependentantibodies or second activation state-independent antibodies and asecond member of the binding pair conjugated to the first member of thesignal amplification pair. The binding pair members (e.g.,biotin/streptavidin) can be coupled to the signal amplification pairmember or to the activation state-dependent antibodies or secondactivation state-independent antibodies using methods well-known in theart. Examples of signal amplification pair members include, but are notlimited to, peroxidases such horseradish peroxidase (HRP), catalase,chloroperoxidase, cytochrome c peroxidase, eosinophil peroxidase,glutathione peroxidase, lactoperoxidase, myeloperoxidase, thyroidperoxidase, deiodinase, and the like. Other examples of signalamplification pair members include haptens protected by a protectinggroup and enzymes inactivated by thioether linkage to an enzymeinhibitor.

In one example of proximity channeling, the facilitating moiety isglucose oxidase (GO) and the first member of the signal amplificationpair is horseradish peroxidase (HRP). When the GO is contacted with asubstrate such as glucose, it generates an oxidizing agent (i.e.,hydrogen peroxide (H₂O₂)). If the HRP is within channeling proximity tothe GO, the H₂O₂ generated by the GO is channeled to and complexes withthe HRP to form an HRP-H₂O₂ complex, which, in the presence of thesecond member of the signal amplification pair (e.g., a chemiluminescentsubstrate such as luminol or isoluminol or a fluorogenic substrate suchas tyramide (e.g., biotin-tyramide), homovanillic acid, or4-hydroxyphenyl acetic acid), generates an amplified signal. Methods ofusing GO and HRP in a proximity assay are described in, e.g., Langry etal., U.S. Dept. of Energy Report No. UCRL-ID-136797 (1999). Whenbiotin-tyramide is used as the second member of the signal amplificationpair, the HRP-H₂O₂ complex oxidizes the tyramide to generate a reactivetyramide radical that covalently binds nearby nucleophilic residues. Theactivated tyramide is either directly detected or detected upon theaddition of a signal-detecting reagent such as, for example, astreptavidin-labeled fluorophore or a combination of astreptavidin-labeled peroxidase and a chromogenic reagent. Examples offluorophores suitable for use in the present invention include, but arenot limited to, an Alexa Fluor® dye (e.g., Alexa Fluor® 555),fluorescein, fluorescein isothiocyanate (FITC), Oregon Green™;rhodamine, Texas red, tetrarhodamine isothiocynate (TRITC), a CyDye™fluor (e.g., Cy2, Cy3, Cy5), and the like. The streptavidin label can becoupled directly or indirectly to the fluorophore or peroxidase usingmethods well-known in the art. Non-limiting examples of chromogenicreagents suitable for use in the present invention include3,3′,5,5′-tetramethylbenzidine (TMB), 3,3′-diaminobenzidine (DAB),2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS),4-chloro-1-napthol (4CN), and/or porphyrinogen.

In another example of proximity channeling, the facilitating moiety is aphotosensitizer and the first member of the signal amplification pair isa large molecule labeled with multiple haptens that are protected withprotecting groups that prevent binding of the haptens to a specificbinding partner (e.g., ligand, antibody, etc.). For example, the signalamplification pair member can be a dextran molecule labeled withprotected biotin, coumarin, and/or fluorescein molecules. Suitableprotecting groups include, but are not limited to, phenoxy-, analino-,olefin-, thioether-, and selenoether-protecting groups. Additionalphotosensitizers and protected hapten molecules suitable for use in theproximity assays of the present invention are described in U.S. Pat. No.5,807,675. When the photosensitizer is excited with light, it generatesan oxidizing agent (i.e., singlet oxygen). If the hapten molecules arewithin channeling proximity to the photosensitizer, the singlet oxygengenerated by the photosensitizer is channeled to and reacts withthioethers on the protecting groups of the haptens to yield carbonylgroups (ketones or aldehydes) and sulphinic acid, releasing theprotecting groups from the haptens. The unprotected haptens are thenavailable to specifically bind to the second member of the signalamplification pair (e.g., a specific binding partner that can generate adetectable signal). For example, when the hapten is biotin, the specificbinding partner can be an enzyme-labeled streptavidin. Exemplary enzymesinclude alkaline phosphatase, β-galactosidase, HRP, etc. After washingto remove unbound reagents, the detectable signal can be generated byadding a detectable (e.g., fluorescent, chemiluminescent, chromogenic,etc.) substrate of the enzyme and detected using suitable methods andinstrumentation known in the art. Alternatively, the detectable signalcan be amplified using tyramide signal amplification and the activatedtyramide either directly detected or detected upon the addition of asignal-detecting reagent as described above.

In yet another example of proximity channeling, the facilitating moietyis a photosensitizer and the first member of the signal amplificationpair is an enzyme-inhibitor complex. The enzyme and inhibitor (e.g.,phosphonic acid-labeled dextran) are linked together by a cleavablelinker (e.g., thioether). When the photosensitizer is excited withlight, it generates an oxidizing agent (i.e., singlet oxygen). If theenzyme-inhibitor complex is within channeling proximity to thephotosensitizer, the singlet oxygen generated by the photosensitizer ischanneled to and reacts with the cleavable linker, releasing theinhibitor from the enzyme, thereby activating the enzyme. An enzymesubstrate is added to generate a detectable signal, or alternatively, anamplification reagent is added to generate an amplified signal.

In a further example of proximity channeling, the facilitating moiety isHRP, the first member of the signal amplification pair is a protectedhapten or an enzyme-inhibitor complex as described above, and theprotecting groups comprise p-alkoxy phenol. The addition ofphenylenediamine and H₂O₂ generates a reactive phenylene diimine whichchannels to the protected hapten or the enzyme-inhibitor complex andreacts with p-alkoxy phenol protecting groups to yield exposed haptensor a reactive enzyme. The amplified signal is generated and detected asdescribed above (see, e.g., U.S. Pat. Nos. 5,532,138 and 5,445,944).

An exemplary protocol for performing the proximity assays describedherein is provided in Example 4 of PCT Publication No. WO2009/108637,the disclosure of which is herein incorporated by reference in itsentirety for all purposes.

In another embodiment, the present invention provides kits forperforming the proximity assays described above comprising: (a) adilution series of one or a plurality of capture antibodies restrainedon a solid support; and (b) one or a plurality of detection antibodies(e.g., a combination of activation state-independent antibodies andactivation state-dependent antibodies for detecting activation levelsand/or a combination of first and second activation state-independentantibodies for detecting expression levels). In some instances, the kitscan further contain instructions for methods of using the kit to detectthe expression and/or activation status of one or a plurality of signaltransduction molecules of cells such as tumor cells. The kits may alsocontain any of the additional reagents described above with respect toperforming the specific methods of the present invention such as, forexample, first and second members of the signal amplification pair,tyramide signal amplification reagents, substrates for the facilitatingmoiety, wash buffers, etc.

VII. Production of Antibodies

The generation and selection of antibodies not already commerciallyavailable for analyzing the levels of expression and activation ofsignal transduction molecules in tumor cells in accordance with theimmunoassays of the present invention can be accomplished several ways.For example, one way is to express and/or purify a polypeptide ofinterest (i.e., antigen) using protein expression and purificationmethods known in the art, while another way is to synthesize thepolypeptide of interest using solid phase peptide synthesis methodsknown in the art. See, e.g., Guide to Protein Purification, Murray P.Deutcher, ed., Meth. Enzymol., Vol. 182 (1990); Solid Phase PeptideSynthesis, Greg B. Fields, ed., Meth. Enzymol., Vol. 289 (1997); Kiso etal., Chem. Pharm. Bull., 38:1192-99 (1990); Mostafavi et al., Biomed.Pept. Proteins Nucleic Acids, 1:255-60, (1995); and Fujiwara et al.,Chem. Pharm. Bull., 44:1326-31 (1996). The purified or synthesizedpolypeptide can then be injected, for example, into mice or rabbits, togenerate polyclonal or monoclonal antibodies. One skilled in the artwill recognize that many procedures are available for the production ofantibodies, for example, as described in Antibodies, A LaboratoryManual, Harlow and Lane, Eds., Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y. (1988). One skilled in the art will also appreciatethat binding fragments or Fab fragments which mimic (e.g., retain thefunctional binding regions of) antibodies can also be prepared fromgenetic information by various procedures. See, e.g., AntibodyEngineering: A Practical Approach, Borrebaeck, Ed., Oxford UniversityPress, Oxford (1995); and Huse et al., J. Immunol., 149:3914-3920(1992).

Those skilled in the art will recognize that many approaches can betaken in producing antibodies or binding fragments and screening andselecting for affinity and specificity for the various polypeptides ofinterest, but these approaches do not change the scope of the presentinvention.

A more detailed description of polyclonal antibodies, monoclonalantibodies, humanized antibodies, human antibodies, bispecificantibodies, fragments thereof, and methods of purifying antibodies isfound in PCT Publication No. WO 2010/132723, the disclosure of which isherein incorporated by reference in its entirety for all purposes.

VIII. Methods of Administration

According to the methods of the present invention, the anticancer drugsdescribed herein are administered to a subject by any convenient meansknown in the art. The methods of the present invention can be used toselect a suitable anticancer drug or combination of anticancer drugs forthe treatment of a tumor, e.g., a breast tumor such as a triple-negativemetastatic breast tumor, in a subject. The methods of the presentinvention can also be used to predict the response of a tumor, e.g., abreast tumor such as a triple-negative metastatic breast tumor, totreatment with an anticancer drug or combination of anticancer drugs. Inaddition, the methods of the present invention can be used to determinethe sensitivity of a tumor cell such as a triple-negative tumor cell,e.g., from a triple-negative metastatic breast tumor, to treatment withan anticancer drug or combination of anticancer drugs. One skilled inthe art will appreciate that the anticancer drugs described herein canbe administered alone or as part of a combined therapeutic approach withconventional chemotherapy, radiotherapy, hormonal therapy,immunotherapy, and/or surgery.

In certain embodiments, the anticancer drug comprises an anti-signalingagent (i.e., a cytostatic drug) such as a monoclonal antibody or atyrosine kinase inhibitor; an anti-proliferative agent; achemotherapeutic agent (i.e., a cytotoxic drug); a hormonal therapeuticagent; a radiotherapeutic agent; a vaccine; and/or any other compoundwith the ability to reduce or abrogate the uncontrolled growth ofaberrant cells such as cancerous cells. In some embodiments, the subjectis treated with one or more anti-signaling agents, anti-proliferativeagents, and/or hormonal therapeutic agents in combination with at leastone chemotherapeutic agent. Exemplary monoclonal antibodies, tyrosinekinase inhibitors, anti-proliferative agents, chemotherapeutic agents,hormonal therapeutic agents, radiotherapeutic agents, and vaccines aredescribed above.

In preferred embodiments, the anticancer drug is a combination ofbevacizumab (Avastin®), carboplatin, and paclitaxel (“triplet therapy”).In some instances, the paclitaxel is a nanoparticle albumin-bound (nab)paclitaxel (Abraxane® or nabP). In other embodiments, the anticancerdrug comprises iniparib (BSI 201; 4-iodo-3-nitrobenzamide), NK012 (anSN-38-releasing nanodevice constructed by covalently attaching SN-38 tothe block copolymer PEG-PGlu, followed by self-assembly of amphiphilicblock copolymers in aqueous media), glembatumumab vedotin, (also knownas CDX-011 or CR011-vcMMAE; human monoclonal antibody glembatumumab(CR011) linked to monomethyl auristatin E (MMAE) that targets cancercells expressing transmembrane glycoprotein NMB), or combinationsthereof. In one particular embodiment, the anticancer drug is acombination of iniparib (a PARP inhibitor), gemcitabine (Gemzar®), andcarboplatin.

In some embodiments, the anticancer drugs described herein can beco-administered with conventional immunotherapeutic agents including,but not limited to, immunostimulants (e.g., Bacillus Calmette-Guérin(BCG), levamisole, interleukin-2, alpha-interferon, etc.), immunotoxins(e.g., anti-CD33 monoclonal antibody-calicheamicin conjugate, anti-CD22monoclonal antibody-pseudomonas exotoxin conjugate, etc.), andradioimmunotherapy (e.g., anti-CD20 monoclonal antibody conjugated to¹¹¹In, ⁹⁰Y, or ¹³¹I, etc.).

Anticancer drugs can be administered with a suitable pharmaceuticalexcipient as necessary and can be carried out via any of the acceptedmodes of administration. Thus, administration can be, for example, oral,buccal, sublingual, gingival, palatal, intravenous, topical,subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint,parenteral, intra-arteriole, intradermal, intraventricular,intracranial, intraperitoneal, intravesical, intrathecal, intralesional,intranasal, rectal, vaginal, or by inhalation. By “co-administer” it ismeant that an anticancer drug is administered at the same time, justprior to, or just after the administration of a second drug (e.g.,another anticancer drug, a drug useful for reducing the side-effectsassociated with anticancer drug therapy, a radiotherapeutic agent, ahormonal therapeutic agent, an immunotherapeutic agent, etc.).

A therapeutically effective amount of an anticancer drug may beadministered repeatedly, e.g., at least 2, 3, 4, 5, 6, 7, 8, or moretimes, or the dose may be administered by continuous infusion. The dosemay take the form of solid, semi-solid, lyophilized powder, or liquiddosage forms, such as, for example, tablets, pills, pellets, capsules,powders, solutions, suspensions, emulsions, suppositories, retentionenemas, creams, ointments, lotions, gels, aerosols, foams, or the like,preferably in unit dosage forms suitable for simple administration ofprecise dosages.

As used herein, the term “unit dosage form” refers to physicallydiscrete units suitable as unitary dosages for human subjects and othermammals, each unit containing a predetermined quantity of an anticancerdrug calculated to produce the desired onset, tolerability, and/ortherapeutic effects, in association with a suitable pharmaceuticalexcipient (e.g., an ampoule). In addition, more concentrated dosageforms may be prepared, from which the more dilute unit dosage forms maythen be produced. The more concentrated dosage forms thus will containsubstantially more than, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more times the amount of the anticancer drug.

Methods for preparing such dosage forms are known to those skilled inthe art (see, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES, 18TH ED., MackPublishing Co., Easton, Pa. (1990)). The dosage forms typically includea conventional pharmaceutical carrier or excipient and may additionallyinclude other medicinal agents, carriers, adjuvants, diluents, tissuepermeation enhancers, solubilizers, and the like. Appropriate excipientscan be tailored to the particular dosage form and route ofadministration by methods well known in the art (see, e.g., REMINGTON'SPHARMACEUTICAL SCIENCES, supra).

Examples of suitable excipients include, but are not limited to,lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia,calcium phosphate, alginates, tragacanth, gelatin, calcium silicate,microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water,saline, syrup, methylcellulose, ethylcellulose,hydroxypropylmethylcellulose, and polyacrylic acids such as Carbopols,e.g., Carbopol 941, Carbopol 980, Carbopol 981, etc. The dosage formscan additionally include lubricating agents such as talc, magnesiumstearate, and mineral oil; wetting agents; emulsifying agents;suspending agents; preserving agents such as methyl-, ethyl-, andpropyl-hydroxy-benzoates (i.e., the parabens); pH adjusting agents suchas inorganic and organic acids and bases; sweetening agents; andflavoring agents. The dosage forms may also comprise biodegradablepolymer beads, dextran, and cyclodextrin inclusion complexes.

For oral administration, the therapeutically effective dose can be inthe form of tablets, capsules, emulsions, suspensions, solutions,syrups, sprays, lozenges, powders, and sustained-release formulations.Suitable excipients for oral administration include pharmaceuticalgrades of mannitol, lactose, starch, magnesium stearate, sodiumsaccharine, talcum, cellulose, glucose, gelatin, sucrose, magnesiumcarbonate, and the like.

In some embodiments, the therapeutically effective dose takes the formof a pill, tablet, or capsule, and thus, the dosage form can contain,along with an anticancer drug, any of the following: a diluent such aslactose, sucrose, dicalcium phosphate, and the like; a disintegrant suchas starch or derivatives thereof; a lubricant such as magnesium stearateand the like; and a binder such a starch, gum acacia,polyvinylpyrrolidone, gelatin, cellulose and derivatives thereof. Ananticancer drug can also be formulated into a suppository disposed, forexample, in a polyethylene glycol (PEG) carrier.

Liquid dosage forms can be prepared by dissolving or dispersing ananticancer drug and optionally one or more pharmaceutically acceptableadjuvants in a carrier such as, for example, aqueous saline (e.g., 0.9%w/v sodium chloride), aqueous dextrose, glycerol, ethanol, and the like,to form a solution or suspension, e.g., for oral, topical, orintravenous administration. An anticancer drug can also be formulatedinto a retention enema.

For topical administration, the therapeutically effective dose can be inthe form of emulsions, lotions, gels, foams, creams, jellies, solutions,suspensions, ointments, and transdermal patches. For administration byinhalation, an anticancer drug can be delivered as a dry powder or inliquid form via a nebulizer. For parenteral administration, thetherapeutically effective dose can be in the form of sterile injectablesolutions and sterile packaged powders. Preferably, injectable solutionsare formulated at a pH of from about 4.5 to about 7.5.

The therapeutically effective dose can also be provided in a lyophilizedform. Such dosage forms may include a buffer, e.g., bicarbonate, forreconstitution prior to administration, or the buffer may be included inthe lyophilized dosage form for reconstitution with, e.g., water. Thelyophilized dosage form may further comprise a suitable vasoconstrictor,e.g., epinephrine. The lyophilized dosage form can be provided in asyringe, optionally packaged in combination with the buffer forreconstitution, such that the reconstituted dosage form can beimmediately administered to a subject.

A subject can also be monitored at periodic time intervals to assess theefficacy of a certain therapeutic regimen. For example, the expressionand/or activation levels of certain signal transduction molecules maychange based on the therapeutic effect of treatment with one or more ofthe anticancer drugs described herein. The subject can be monitored toassess response and understand the effects of certain drugs ortreatments in an individualized approach. Additionally, subjects whoinitially respond to a specific anticancer drug or combination ofanticancer drugs may become refractory to the drug or drug combination,indicating that these subjects have developed acquired drug resistance.These subjects can be discontinued on their current therapy and analternative treatment prescribed in accordance with the methods of thepresent invention.

In certain aspects, the methods described herein can be used inconjunction with panels of gene expression markers that predict thelikelihood of breast cancer prognosis and/or recurrence in variouspopulations. These gene panels can be useful for identifying individualswho are unlikely to experience recurrence and, thus, unlikely to benefitfrom adjuvant chemotherapy. The expression panels can be used toidentify individuals who can safely avoid adjuvant chemotherapy, withoutnegatively affecting disease-free and overall survival outcomes.Suitable systems include, but are not limited to, Oncotype DX™, which isa 21-gene panel from Genomic Health, Inc.; MammaPrint,® which is a70-gene panel from Agendia; and a 76-gene panel from Veridex.

In addition, in certain other aspects, the methods described herein canbe used in conjunction with panels of gene expression markers thatidentify the original tumors for cancers of unknown primary (CUP). Thesegene panels can be useful in identifying patients with metastatic cancerwho would benefit from therapy consistent with that given to patientsdiagnosed initially with breast cancer. Suitable systems include, butare not limited to, the Aviara CancerTYPE ID assay, an RT-PCR-basedexpression assay that measures 92 genes to identify the primary site oforigin for 39 tumor types; and the Pathwork® Tissue of Origin Test,which measures the expression of more than 1600 genes on a microarrayand compares a tumor's gene expression “signature” against those of 15known tissue types.”

IX. Examples

The following examples are offered to illustrate, but not to limit, theclaimed invention.

The Examples from PCT Publication No. WO 2010/132723 are hereinincorporated by reference in their entirety for all purposes.

Example 1 Exemplary Proximity Assay Slide Format

This example illustrates one preferred embodiment of the proximityassays of the invention, also known as the Collaborative Enzyme EnhancedReactive ImmunoAssay (CEER). The proximity assays of this embodiment usean antibody-microarray based platform that measures the expression andactivation of target proteins in circulating tumor cells (CTCs) and/ortissue samples (e.g., FNAs). As a non-limiting example, the proximityassays of this embodiment can be used to analyze the level of proteinexpression and/or the status of activation of one or more targetproteins such as HER1 in CTCs or tumor tissue. In some instances, theproximity assays of this embodiment utilize CTCs isolated from about 7.5ml of whole blood by magnetic particles coated with anti-Ep-CAMantibodies using the CTC-Profiler (Veridex). Isolated CTCs may then bestimulated with growth factors (e.g., EGF+Heregulin) prior toimmuno-analysis of subsequent ErbB pathway expression and/or activation.In other instances, the proximity assays of this embodiment utilizetumor tissue samples including fresh frozen metastatic biopsies such as,e.g., triple-negative breast cancer samples.

In certain instances, the proximity assays of this embodiment use aslide format and include multiple calibrators and controls. FIG. 1 showsthe array designs of exemplary slide formats for analyzing total andphosphorylated HER1 and HER2 levels. There are 16 pads on each slidewith room for 300 spots on each pad. A contact microarray printer wasused to print on the 16 pad nitrocellulose slides. Each spot includes atracking dye and either a specific capture antibody (Ab) or controlsprinted in triplicates in serial dilutions. The capture Abs are printedat 1 mg/ml, 0.5 mg/ml, and 0.25 mg/ml. Purified IgG was printed as anorientation reference in both the Total and Phospho assays. BSA-phosphowas printed as a reagent control. Analytical calibration reactions areperformed on 8 pads and internal quality control reactions on 2 pads.Each slide allows processing of up to 4 unknown patient samples.Expression of total target proteins or phosphorylated activated proteinscan be reported in Computed Unit (CU), a unit based on calculation fromstandard curves of diluted lysate from positive cell lines which expressthe protein of interest. Two separate slides are used for each sample;one slide to detect the expression of the target proteins in cellsisolated from whole blood (“Total Assay Slide”) and the other for thedetection of phosphorylation to detect the degree of target proteinactivation (“Phospho Assay Slide”).

In one embodiment, whole blood from patients and normal controlindividuals are collected in EDTA tubes. In order to prevent any skincell contamination during blood draw, our procedures stipulate that thefirst 3 mL of blood collected is discarded (or collected in CellSavetube for CTC counts and visual immuno-staining using CellSearch kit).Two additional EDTA tubes are then used to collect 7.5 mL of whole bloodin each tube. CTCs are then isolated from each tube using an automatedmagnetic cell separation device (Veridex AutoPrep). Enriched samples arecombined and then stimulated with growth factors. Activated cells arethen lysed and either immediately processed or stored at −80° C. forsubsequent immuno-analysis. In another embodiment, cells from tumortissue such as fresh frozen metastatic biopsies are obtained, lysed toproduce a cellular extract, and then either immediately processed orstored at −80° C. for subsequent immuno-analysis.

The proximity assays of this embodiment are initiated by incubatingprotein targets in cell lysates with capture antibodies on animmuno-microarray surface. Any HER1 or other RTK or signal transductionpathway protein in cell lysates are bound to their corresponding captureantibodies and subsequently unique immuno-complexes are formed by twoadditional detector antibodies. One of the detector antibodies isconjugated to glucose oxidase (GO) and generates H₂O₂ in the presence ofglucose. When the second HRP-conjugated detector antibody is bound inproximity within the immuno-complex, a positive signal is generated. Thesubsequent tyramide-mediated signal amplification process enhances thesensitivity of the assay. The specificity of protein detection isenhanced by the concurrent binding of three specific Abs to differentepitopes, and sensitivity can be as high as a single cell due to theamplification. FIG. 2 shows a schematic of an exemplary proximity assayfor detecting phosphorylated HER1.

The microarray platform described herein offers the benefit ofmultiplexing. The ability to expand the assay enables high contentanalysis with the measurement of multiple receptors and signalingmolecules from limited available sample. The microarray is scalable andhas the potential for achieving the throughput needed for a clinicallyuseful diagnostic assay.

Example 2 EGFR and VEGFR2Expression Predict Response to Nab-paclitaxel(nabP)/Carboplatin (C)/Bevacizumab (B) Chemotherapy in Triple-NegativeMetastatic Breast Cancer (TNMBC)

Background

Patients with triple-negative metastatic breast cancer (TNMBC), in whichtheir cancer demonstrates no expression of estrogen, progesterone, orhuman epidermal growth factor receptor 2 (HER2) receptors, face a poorprognosis (Dent, R. et al., Clin Cancer Res. 13(15 Pt 1):4429-4434(2007)). Improved therapies and predictive markers are needed in thissetting.

B (bevacizumab; Avastin®) and C (Carboplatin) combinations are active inmany solid tumors, including TNMBC. Nab-paclitaxel (nabP; Abraxane®)potentially targets SPARC, an albumin-binding protein secreted bytumors.

Taxane- and platinum-based chemotherapies have significant activity inthe first-line treatment of metastatic breast cancer and areparticularly effective when given in combination (Perez, E. A.,Oncologist 9(5):518-527 (2004); Perez, E. A. et al., Oncology69(2):117-121 (2005); O'Shaughnessy J., Oncologist 10(Suppl 3):20-29(2005)).

Nanoparticle albumin-bound (nab)-paclitaxel has improved efficacy andtolerability compared with standard paclitaxel (Gradishar, W. J. et al.,J Clin Oncol. 23(31):7794-7803 (2005)).

Among the platinum agents, carboplatin appears to have similar efficacyand is better tolerated than cisplatin when combined with a taxane, witha lower risk of non-hematologic toxicity, although with a somewhatgreater risk of hematologic toxicity (Gainford, C. et al., Proc Am SocClin Oncol. 19:113, Abstract 439 (2000); Perez, E. A. et al., Cancer88(1):124-131 (2000); Perez, E. A. et al., Oncology 69(2):117-121(2005)). The incidence of neutropenia lessens when carboplatin iscombined with paclitaxel rather than docetaxel (Perez, E. A. et al.,Cancer 88(1):124-131 (2000); Perez, E. A. et al., Oncology 69(2):117-121(2005)).

Bevacizumab is a monoclonal antibody that targets vascular endothelialgrowth factor (VEGF) to inhibit angiogenesis. Adding bevacizumab totaxane-based chemotherapy significantly improves treatment responserates and progression-free survival (PFS) in women with metastaticbreast cancer (Miller, K. D. et al., N Engl J Med. 357(26):2666-2676(2007); Miles, D. W. et al., Cancer Res. 69(24S):495s, Abstract 41(2009); Robert, N. J. et al., J Clin Oncol. 27(15 suppl):42s, Abstract1005 (2009)).

Objective

This study used enrollment metastatic biopsies (bxs) of untreated TNMBCto (1) describe expression/activation of signaling pathways in TNMBC,and (2) correlate these expression patterns in TNMBC with response.

Methods

Fresh frozen metastatic biopsies obtained at trial initiation were usedto assess the expression and activation of signaling pathway proteins(e.g., HER1 (EGFR), HER2, HER3, insulin-like growth factor receptor 1(IGF1-R), c-KIT, c-MET, PI3K, AKT, MAPK, and/or VEGFR2). A proximityassay platform such as CEER was used to determine comprehensive pathwayprotein expression and activation. The log-rank test was used to testthe association between progression free survival (PFS) and proteinexpression or activation of the pathway proteins. The tumor tissuesamples used in this study were obtained from an ongoing clinical trialentitled “A Phase II Study of Abraxane®, Carboplatin and Bevacizumab[triplet therapy] in ‘Triple Negative’ (Demonstrating No Expression forEstrogen, Progesterone, or Her2 Receptors) Metastatic Breast Cancer”with the following ClinicalTrials.gov identifier: NCT00479674, thedisclosure of which is herein incorporated by reference in its entiretyfor all purposes.

Results

Total EGFR was expressed in 11/18 (61%) biopsies, but was activated in3/18 (17%). PI3K and AKT were overexpressed in 11/18 (61%) and 8/18(44%) patients, respectively, and were co-expressed in 6/18 (33%)biopsies. In 17 metastatic biopsies, there was higher total EGFRexpression (33 wks vs. 22 wks, p=0.14) and lower expression of VEGFR2(33 wks vs. 18 wks, p=0.16). All measurements were performed by CEER.

Conclusions

Nab-Paclitaxel/Carboplatin/Bevacuzimab offers a well tolerated andeffective therapy for TNMBC. Overexpression of total EGFR and lowerVEGFR2 levels offer predictive value for response to triplet therapy inTNMBC. Real-time metastatic biopsies indicate that important changesoccur between primary and metastatic tumors, and these changes arehighly influential in defining predictive markers of response for TNMBC.

Example 3 Comprehensive Pathway Profiling to Predict Response to TherapyContaining Carboplatin (C)/Bevacizumab(B) in Triple-Negative MetastaticBreast Cancer (TNMBC)

Background: Improved therapies and predictive markers are needed inTNMBC. B (Avastin™) and C (Carboplatin) combinations are active in manysolid tumors, including TNBC. This study used archival primary andmetastatic biopsies (bxs) of untreated TNMBC (1) to determine the levelof expression of VEGFR2 in primary and TNMBC, (2) to describeexpression/activation of various signaling pathway proteins in TNMBC,and (3) to correlate these expression patterns in TNMBC with response.

Methods: Triple negative breast cancer core-biopsy specimens werecollected and appropriately frozen at −80° C. A novel immunoassay methodwas applied to investigate the levels of expression and activation ofsignaling proteins in 1000 ng to 5000 ng of frozen tissues. The CEERplatform (aka COPIA) is a multiplexed immuno-microarray platform thatutilizes the formation of a unique immuno-complex requiring theco-localization of two detector-antibodies for channeling events forsignal generation/amplification resulting in extremely high analyticalsensitivity and specificity.

Results: This study identified varying degrees of CK values in eachspecimen (from approximately 3 to 1000 tumor cells/1000 ng to 5000 ng ofcell lysate). In these TNMBCs, HER2 expression levels ranged from low tomoderate (0 to 2+, IHC equivalent) and were found in 17 out of 18samples. One out of 18 samples had substantially high levels of HER2expression (IHC 3+ level). 50% of the specimens did not show HER2phosphorylation while the other 50% showed varying levels of activatedHER2. The prevalence of HER1, HER3, IGF1-R, c-KIT, c-MET, PI3K, Shc,VEGFR2, and AKT expression/activation was also analyzed in thesesamples.

Conclusion: Real-time metastatic biopsies indicate that importantchanges occur between primary and metastatic tumors, and these changesare highly influential in defining predictive markers of response.Over-expression of total and activated EGFR, and lower VEGFR2 levelsoffer predictive value for response to triplet therapy(B+C+Nab-paclitaxel) in TNMBC. As disease-profile often shifts,monitoring of alterations in transduction pathway proteins will beuseful for therapy adjustments.

Example 4 Functional Profiling of Multiple Signal Pathway Proteins inBreast Cancer Patients

Abstract

One of the mechanisms of de novo or acquired resistance is theexpression of various forms of truncated HER2/ERBB2 receptors(“t-ERBB2”) with missing amino-terminal extracellular domains.Non-limiting examples of t-ERBB2 isoforms include p110, p95HER2 (p95m),p95c, and p95n. Methods for profiling various forms of HER2 receptorsand other receptor tyrosine kinases (RTKs) with transactivationpotential in primary and metastatic tumors may provide valuable insightinto the shifting disease pathogenesis. This example describes thesuccessful profiling of a panel of signal transduction pathway proteinsfor their expression and activation in 230 breast cancer with variousER/PR/HER2 status. In particular, the levels of total and phosphorylatedt-ERBB2 species in human breast tumor samples were investigated usingthe novel proximity mediated immuno-microarray method described herein.This example shows that t-ERBB2 isoforms were detected in strongly ERBB2positive tumors (16 of 31 samples, 52%) and were phosphorylated in 10 of38 samples (32%).

Introduction

Several mechanisms for Trastuzumab resistance have been reported.Primarily, the activation of other RTKs (such as IGF1-R) and theaccumulation of truncated forms of HER2 have been frequently reported,among other mechanisms. In particular, the amino terminally truncatedcarboxyl terminal fragments of HER2, collectively known as p95HER2, arefrequently found in HER2-expressing breast cancer cell lines and tumors.Cross-talk between various signal transduction pathways and feedbackloops provide escape mechanisms for tumors under certain therapeuticpressure or pathway addiction and requires a comprehensive diagnostictool to perform “pathway network analysis.” Treatment decisions madebased on clinical information obtained through current IHC/FISH-basedtechnology performed for a few selected biomarkers will not be effectivein treating patients with rapidly evolving heterogeneous disease. Thisexample demonstrates that a different configuration of detectorantibodies allows differential detection of truncated targets (e.g.,p95HER2) from their full-length counterparts (e.g., HER2). Inparticular, this example illustrates the use of a novel, highlysensitive and specific antibody microarray format, Collaborative EnzymeEnhanced Reactive ImmunoAssay (CEER), to quantify the levels of totaland phosphorylated t-ERBB2 species in human samples, in both flashfrozen tissue and fine-needle aspirates of metastatic tumors. Thisexample further demonstrates an analysis of the functional status(expression and activation) of HER2, p95HER2, HER1, HER3, and IGF1R aswell as the downstream signal transduction proteins PI3K, Shc, andc-MET.

Methods

Multiplexed microarray printing: Capture antibodies (Abs) were printedon nitrocellulose-coated glass slides (ONCYTE®, Grace Bio-Labs) usingnon-contact printers (Nanoplotter, GeSiM). The spot diameter wasapproximately 175 μm and printed slides were kept in a desiccatedchamber at 4° C. Each spot included a tracking dye and specific captureAbs. Approximately 500 pL of capture Abs were printed in triplicate andat serial dilution concentrations of 1 mg/mL, 0.5 mg/mL, and 0.25 mg/mL.Purified mouse-IgGs were printed as a negative control. Each slidecontains cell line controls for standard curve generation for accuratequantitation of samples on each slide run. Internal quality controlsamples are run on each slide to ensure the quality of data generatedfrom each array-slide.

Antibody conjugation and purification: Target-specific Abs andcorresponding detector enzymes were activated with a bi-functionalcross-linker,succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), andcoupled to dextran to make antibody-dextran-enzyme polymer conjugates.The conjugate was purified by HPLC using a size-exclusion column. The Abactivities in the purified conjugates were detected by competition ELISAand enzyme activity was detected by a functional assay specific for eachdetector enzyme.

Collaborative Enzyme Enhanced Reactive Immunoassay (CEER): As depictedin FIG. 3, target proteins present in tissue lysates are bound tospecific capture antibodies printed on the nitrocellulose surface andunbound non-target proteins are removed from the slide. The enzymaticinteraction between one detector antibody against an alternate epitopeon a captured target protein conjugated to Glucose Oxidase (GO) and theother detector antibody specific for a phosphorylated site on the targetprotein or another non-overlapping epitope conjugated to HRP results insignal generation and subsequent tyramide-mediated signal amplification.In particular, the immuno-microarray slides were rinsed 2 times withTBST (50 mM Tris/150 mM NaCl/0.1% Tween-20, pH 7.2-7.4), blocked with 80μL of Whatman Blocking Buffer for 1 hr at RT, and then washed 2 timeswith TBST. Serially diluted cell lysate controls in 80 μL of dilutionbuffer (2% BSA/0.1% Triton X-100/TBS, pH 7.2-7.4) and samples were addedto sub-arrays and designated for standards on the slide, then incubatedfor 1 hour at RT. After incubation, slides were washed 4 times, 3 min.each time. The detector Abs were added in 80□μL of the reaction bufferand incubated for 2 hours at RT. After unbound secondary detector Abswere removed by washing with TBST, 80 μL of biotin-tyramide (400 μg/mLin ethanol, Perkin Elmer Life Science) at 5 μg/ml in 50 mM glucose/PBSwas added and incubated for 15 min in the dark. The GO-HRP channeledtyramide-mediated signal amplification process was terminated by washingwith TBST 4 times for 3 min each. The local deposition ofbiotin-tyramide was detected by adding streptavidin (SA)-Alexa647 (inPBS, Invitrogen) at 0.5 μg/ml (1:4000 dilution) in 2% BSA/0.1%Triton/TBS for 40 min. Upon completion of incubation, slides were washed4 times with TBST, dried and kept in the dark until scanning on themicroarray scanner.

tErbB2 (e.g., p95HER2) Enrichment: Full-length p185-ErbB2 receptors wereremoved from cell lysates using magnetically labeled antibodies specificto the extracellular domain (ECD) of ErbB2. The resulting p185-ErbB2depleted cell lysate contains t-ERBB2 receptor proteins lacking the ECDand subsequent analysis were performed to quantitate the level oft-ERBB2 expression and activation.

Clinical Samples: The flash frozen breast cancer tissues were purchasedfrom ILSBio. All patients were Caucasian with ductal carcinoma at stageII or III. ErbB2-IHC status was available for all samples. The flashfrozen tissue samples were lysed in 100 μl of lysis buffer. Lysedsamples were kept on ice for 30 min and centrifuged. The proteinconcentrations of supernatants were determined by BCA protein assay kit(Pierce), and the resulting lysates were stored at −80° C. beforesubsequent analysis. FNA samples were collected from patients withprogressive, measurable metastatic Stage 111B, or Stage 1V breastcancer, and who were about to start systemic therapy. Patients hadhistologically or cytologically confirmed invasive breast cancer. TheFNA samples were collected using G23 gauge needles. FNA samples wereimmediately injected into collection vials containing lysis buffer andwere shipped over night for subsequent analysis.

IP-Western Blotting: The cell lysates were incubated with magnetic beadsconjugated with antibodies against the ICD of ERBB2 overnight on arocker at 4° C. The immuno-magnetically enriched lysates wereresuspended in sample buffer containing β-mercaptoethanol, boiled for 5min, cooled to RT and loaded onto a NuPage (Invitrogen) 4-12% gel. Uponcompletion, the separated proteins were transferred to a nitrocellulosemembrane, then washed, blocked with 5% milk blotto, and incubated withthe 1° then 2° Abs before the detection process using NBT/BCIP.

CEER Data Analysis: Each slide was scanned at three photomultiplier(PMT) gain settings to increase the effective dynamic range.Background-corrected signal intensities were averaged for replicatespots printed in triplicate. The relative fluorescence value of therespective reagent blank was subtracted from each sample. Severalquality criteria were used to filter data from further analysisincluding limits on the spot footprint, coefficient of variation forspot replicates, overall pad background and the intensity of the reagentblank. For each assay, a standard curve was generated from seriallydiluted cell lysates prepared from BT474 cells. Data was fit to a fiveparameter equation derived as a function of capture antibodyconcentration and PMT. Each curve was plotted as a function of logsignal intensity, measured as relative fluorescence unit (RFU) vs. logconcentration and referenced to the standard cell line, BT474. Theindividual predictions from each dilution and gain were averaged into asingle, final prediction.

Results

Expression of t-ERBB2s in Human Cells and Tumors. To assess the levelsand activation of ERBB2 isoforms in human cells and tumor samples, CEER,a novel antibody capture and proximity based immune-microarray platform,was employed (FIG. 3). CEER requires an immuno-complex formation betweenthe target-specific capture antibody and two additional detectorantibodies. One of the detector antibodies is conjugated with GlucoseOxidase (GO) and the other is conjugated with Horseradish Peroxidase(HRP). The detector antibodies can bind to either two different epitopes(which determines the target expression level) or to a phosphorylateddomain on the captured protein and one alternative epitope (whichdetermines the target activation level). When GO in the immune-complexis supplied with a substrate such as glucose, it generates hydrogenperoxide (H₂O₂) which is channeled to the co-localized HRP, therebyenhancing the analytical sensitivity. As the assay configurationrequires a successful immuno-complex formation between multiplecapture/detector antibodies, the platform provides the specificitynecessary for simultaneous analysis on multiple target proteins. Asshown in FIG. 4, this method is able to detect total and phosphorylatedERBB2 in BT474 cell lines with single digit analytical sensitivity. Thecomparison of the differential ERBB2 profiling (with ERBB2-ECD andERBB2-ICD captures) of BT474 cells with and without the removal of thefull-length p185-ERBB2 showed that there was approximately 4.4% t-ERBB2(or ˜52800 tERBB2 receptors/cell) in this ERBB2 amplified cell line withapproximately 1.2×10⁶ ERBB2 receptors/cell (FIG. 5).

Frozen breast tumor samples from 74 patients were scored for ERBB2levels using immunohistochemical analysis and were then analyzed usingCEER (Table 3). Of the 74 samples, 24 were ERBB2 low/negative (score=0-1by IHC), 19 had moderate ERBB2 expression (score=2), and the remaining31 had high expression of ERBB2 (score=3). By CEER analysis, none of theERBB2 low or negative tumors expressed a significant level of truncatedERBB2, as expected. The levels of ERBB2 and t-ERBB2 and phosphorylatedt-ERBB2 in each sample (shown in RTK molecules/cell in reference toBT474 cells) are summarized in the Table 2. However, 10% (2 of 19)moderate ERBB2-positive tumors expressed t-ERBB2, and 52% (16 of 31)strongly ERBB2-positive tumors expressed t-ERBB2. Furthermore, t-ERBB2isoforms were phosphorylated in both moderately ERBB2-positive tumors(10%, 2 of 19 samples) and high-ERBB2 tumors (32%, 10 of 31). There weresamples with significant levels of tERBB2 phosphorylation and onlymoderate levels of t-ERBB2 (20330 and 24913), and this may be possibleas t-ERBB2 can be activated through interaction with other RTKs.Examples of tumor CEER-ERBB2 profiling and IP-Western analysis of ERBB2are provided in FIG. 6 (samples are underlined in Table 2) and FIG. 7(samples are boxed in Table 2), respectively. In addition, FNAs frommetastatic sites of 8 breast cancer patients were analyzed. Threesamples with ERBB2-positive disease showed varying degree of t-ERBB2 andphosphorylated t-ERBB2 (Table 3) while samples from ERBB2 negativecancers did not show t-ERBB2 expression. On expanded RTK profiling,IGF1R and c-MET expression was detected in 8C3-005-006, and this may bethe cause for higher level of pt-ERBB2 despite lower level of t-ERBB2among ERBB2 positive FNA samples.

TABLE 1 Expression and phosphorylation of t-ERBB2 in human breasttumors. IHC ERBB2 score 0/1 ERBB2 score 2 ERBB2 score 3 sample # 24  1931 COPIA t-ERBB2+ 0  2 16 COPIA t-ERBB2-P+ 0  2 10 % t-ERBB2+    0%   11%    52% % t-ERBB2+    0%    11%    32%

TABLE 2 ERBB2 profiling of breast cancer tissue. p185HER2-T tHER2-TtHER2-P Sample IHC [RTK/cell) (RTK/cell) (pRTK/cell) 24315 3 1,253,21131,342 247 20013 3 >2 ×10e6 74,298 150 25066 3 1,876,717 62,646 16226110 3 >2 × 10e6 75,942 1,007

3 >2 × 10e6 314,177 3,565

3 1,108,050 41,569 81

3 >2 ×10e6 52,315 297 20012 3 1,840,292 75,450 1,289

3 1,501,949 154,422 371 20003 3 1,227,092 44,179 566 19730 3 1,753,386126,745 390 OV8S1 3 937,546 25,552 41

3 >2 × 10e6 107,268 6,464 20520 3 >2 × 10e6 113,161 3,687 21704 31,520,673 48,669 93 26811 3 >2 × 10e6 307,337 4,523 26106 3 >2 × 10e6125,062 935 22080 3 1,340,073 142,055 2,215 19844 3 956,628 49,042 15820371 3 >2 × 10e6 105,413 728 AUBBG 3 486,835 15,525 56 22715 31,148,445 58,781 3,467 21703 3 643,236 31,597 92 19927 3 1,452,31391,566 563 20330 3 458,839 45,080 1,251 21657 3 292,283 10,048 28 226103 745,833 39,916 195 24720 3 511,344 28,431 524 26775 3 664,308 36,527397 25058 3 655,416 86,973 5,506 19871 3 223,808 25,221 235 22113 2 >2 ×10e6 66,497 724 ZCCFFAK4 2 433,513 13,746 135 26773 2 946,789 208,28216,141 26780 2 538,384 7,338 —

2 393,028 36,054 2,145 25283 2 118,612 7,000 25

2 — — — 26379 2 221,142 6,468 52 WUQT6 2 314,653 17,864 47 25882 2219,387 3,026 93

2 137,399 10,424 248 24960 2 — 15,563 43 26154 2 110,091 3,511 111 250612 — 8,993 105 22176 2 — 3,788 95 21962 2 — 1,515 102

2 — 4,769 16 20525 2 — 1,806 50 24916 2 — 2,224 15 26814 1 313,244 2,86172 22090 1 291,607 3,481 35 22112 1 — — — 24272 1 116,945 20,619 26519875 1 — 5,448 110 19924 1 — 4,123 15 20014 1 — 10,040 43 26371 1 —13,302 469

1 — — — 24400 1 — 10,078 93 26776 1 — 346 — 19826 1 — 21,840 75 24931 1— 8,054 95 KW7YHAET 1 — 3,367 36 NP11802 1 — 2,053 17 19898 1 — 894 —21655 0 13,941 11,983 65 24676 0 — 2,905 —

0 — 8,896 214 19692 0 — 2,664 50

0 — — — 20007 0 — 5,536 52

0 — 4,463 44 1R2H7 0 — — 66 The exemplary CEER array images are shownfor the underlined samples in FIG. 6 and the IP-Western blot for theboxed samples are shown in FIG. 7. The RTK/cell values are determined bycomparing the input amount of samples and equivalent amount of standardBT474 cells.

TABLE 3 t-ERBB2 analysis for FNA samples. Sample ID t-ERBB2 (RTK/cell)pt-ERBB2 (pRTK/cell) 8C3-002-001 57,341 3,062 8C3-005-006 26,989 5,3098C3-005-007 50,741 5,204

A wide range of pathway protein expression and activation in 174 BCAsamples was observed as shown in FIG. 8. The sample with the highestsignal for each marker is indicated with the darkest color. TheCEER-HER2 showed high correlation with IHC-HER2 status. Discordant HER2status between CEER and IHC was resolved by IP-Western and showed >98%correlation. The HER3-P level showed high degree of correlation withHER3-T and PI3K activation. The cMET profile also showed strongcorrelation with PI3K activation. 27% (12/45 of 2+ by IHC) and 21%(11/53 of 0/1+ by IHC) of BCA tissues with non-overexpressing but withsignificant levels of HER2 showed over 5% phosphorylation of expressedHER2 receptor.

Conclusion

The status of HER2 and its variant forms as well as other RTKs providescritical information on the potential mechanisms for HER2-positive BCApatients who do not respond to trastuzumab due to either primary oracquired resistance. The CEER analysis described herein can be utilizedto profile BCA patients for their signal transduction proteins forselecting an effective targeted therapy.

Example 5 Comprehensive Pathway Profiling to Predict Response to Therapyin Triple-Negative Metastatic Breast Cancer (TNMBC)

Improved therapies and predictive markers are needed in TNBC. This studyused core-biopsy specimens from triple negative breast cancer patientstreated with B (Avastin® [bevacizumab]), C (Carboplatin), and nabP(Abraxane®) (“triplet therapy”) to (1) determine the level of expressionand activation of various signaling pathway proteins in TNMBC (e.g.,VEGFR2, c-KIT, HER1, etc.) and (2) correlate these expression andactivation patterns in TNMBC with response.

Triple negative breast cancer core-biopsy samples (n=17) obtained frompatients prior to starting treatment with B (Avastin® [bevacizumab]), C(Carboplatin), and nabP (Abraxane®) were collected and appropriatelyfrozen at −80° C. A novel immunoassay method was applied to investigatethe levels of expression and activation of signaling proteins in 1000 ngto 5000 ng of frozen tissues. The Collaborative Enzyme Enhanced ReactiveImmunoAssay (CEER) (also known as the COllaborative ProximityImmunoAssay (COPIA)) as described herein is a multiplexedimmuno-microarray platform that utilizes the formation of a uniqueimmuno-complex requiring the co-localization of two detector antibodiesfor channeling events to achieve signal generation/amplification thatresults in extremely high analytical sensitivity and specificity. FIG. 9provides an example of functional pathway profiling by CEER on atriple-negative breast cancer core-biopsy sample compared to controlT47D breast cancer cells and human umbilical vein endothelial cells(HUVEC). In particular, the expression and activation of the followingsignaling pathway proteins in TNMBC were evaluated by CEER: EGFR (HER1),HER2, HER3, c-Met, IGF-1R, c-KIT, PI3K, SHC, AKT, and VEGFR2.

Progression Free Survival (PFS) was also determined. For each marker,samples were split into “high” and “low” groups based on a cutoff value.For example, the cutoff value can be selected at or close to the medianto equalize group sizes (e.g., 8=low, 9=high). The parametric t-testtest was used to correlate protein expression and activation with PFS.The PFS was compared for the low (e.g., below median) versus high (e.g.,above median) groups. FIG. 10 illustrates the results of such acomparison between the PFS for the low and high sample groups for eachmarker. In particular, the table in FIG. 10 shows that low c-KIT or lowVEGFR2 expression is associated with a significantly longer duration ofPFS compared to higher levels of that marker (42.1 weeks PFS for lowtotal c-KIT levels versus 20.7 weeks PFS for high total c-KIT levels;39.9 weeks PFS for low total VEGFR2 levels versus 20.9 weeks PFS forhigh total VEGFR2 levels). The table in FIG. 10 also shows that lowIGF-1R activation, low c-KIT activation, low IGF-1R expression, and lowHER1 activation were each associated with longer duration of PFScompared to higher levels of that marker. In addition, the table in FIG.10 shows that high HER1 expression is associated with a longer durationof PFS compared to lower levels of that marker (36.8 weeks PFS for hightotal HER1 levels versus 23.4 weeks PFS for low total HER1 levels). Thetable in FIG. 11 illustrates a similar analysis using a nonparametricWilcoxon rank sum test to correlate protein expression and activationwith PFS. Low c-KIT or VEGFR2 expression was again shown to beassociated with a significantly longer duration of PFS compared tohigher levels of that marker.

FIG. 12 shows that measuring the expression levels of both c-KIT andVEGFR2 increases the predictive value of determining response to triplettherapy in TNMBC. In particular, a combination of low c-KIT and VEGFR2expression levels (“Neither high”) was found to be associated with asignificantly longer duration of PFS compared to samples in which one orboth markers were high (“One high” or “Both high”).

FIG. 13 shows that measuring the expression levels of both VEGFR2 andHER1 increases the predictive value of determining response to triplettherapy in TNMBC. In particular, a combination of high HER1 and lowVEGFR2 expression levels (“Neither bad”) was found to be associated witha significantly longer duration of PFS compared to samples in which HER1expression was low and/or VEGFR2 expression was high (“One bad” or “Bothbad”).

FIG. 14 illustrates the correlation between increasing levels of (A)total VEGFR2, (B) total c-KIT, and (C) total HER1 and response totriplet therapy, as indicated by the length of PFS. In particular, lowertotal VEGFR2 or c-KIT levels were associated with response to triplettherapy in TNMBC, whereas higher total HER1 levels were associated withresponse to triplet therapy in TNMBC.

In conclusion, this example demonstrates that: (1) there is acorrelation between activated (phospho) and total levels for both c-KITand IGF-1R; (2) low VEGFR2 or c-KIT expression levels are predictive ofresponse; (3) high EGFR (e.g., should respond to platinum basedtherapies) expression levels correlate with response, while high P-EGFRlevels do not; (4) in general, if pathway activation (e.g., P-c-KIT,p-IGF-1R, p-EGFR, p-AKT, and others) is low (e.g., not highlyproliferative tumor), the patient has a higher PFS to triplet therapy ofCarboplatin, Nab-Pactitaxel, and Avastin; and (5) for combinationtherapeutics, the addition of total VEGFR2 expression to total c-KIT orEGFR expression results in better predictive values.

Example 6 Tumor Tissue Collection and Processing

Tumor tissue or FNA samples may be obtained from patients with cancerfor signal transduction pathway interrogation using the proximity assaysdescribed herein (e.g., CEER). For example, samples for pathway analysiscan be obtained from frozen tissues either by sectioning or performing afrozen FNA procedure. In certain instances, tissue sectioning isperformed on frozen specimens for subsequent profile analysis. Incertain other instances, the relatively non-invasive FNA procedure isperformed for obtaining samples from patients (and xenografts) in aclinical environment.

Frozen tissue samples may be collected by the following methods:

Option #1. Tissue Section Collection:

-   -   1. Keep a plastic weighing boat on dry ice, in which sample        cutting will take place.        -   a. To chill the materials, keep razor blades or microtome            blades, fine forceps, and pre-labeled sample collection            vials on dry ice.    -   2. Take frozen human cancer tissues from −80° C. freezer and        transfer samples immediately onto dry ice.    -   3. Place frozen tissue to weighting boat on dry ice, cut small        pieces of frozen tissue (10 μm section×3) using razor blade or        microtome blade, and transfer the tissue into pre-chilled and        pre-labeled sample collection vial using pre-chilled forceps.    -   4. Close cap and keep it on dry ice.    -   5. Place collected specimens into a double plastic bag first and        then into a STYROFOAM™ container (primary container) with        adequate amount of dry ice.        -   a. Use at least 6-8 pounds dry ice. Use more in the summer            months.            -   NOTE: Exact amount of dry ice will be determined after                consulting with a shipping company.        -   b. Consult with shipping company for the international            shipping process for necessary permits and documentations.        -   c. Do not use wet ice, or coolants (i.e., Cool Packs).    -   6. Make certain the requisition and sample list is placed in the        box, but on the outside of the double bag.    -   7. Securely seal the container and label “Frozen Tissue-Do Not        Thaw.”        Option #2. FNA Prep from Frozen Tissues:    -   1. Take frozen human cancer tissues from −80° C. freezer and        transfer sample vials immediately on dry ice.    -   2. Samples ready for FNA procedure should be placed on wet ice        for 10 minutes to soften the tissue.    -   3. FNA sample collection should be performed by passing a 23 or        25 gauge needle through softened frozen tissue 5 to 10 times.        Return remaining sample vial to dry ice.    -   4. Wipe the FNA sample collection vial lid with alcohol.    -   5. Frozen FNA tissues should be collected by direct injection        into the collection vial containing 100 μl of “protein later        solution” (Prometheus Laboratories; San Diego, CA). Dispense        collected tissue materials by gently mixing the content.    -   6. Hold the FNA collection vial firmly with one hand and perform        rapid finger tapping (˜15×) to ensure through cell lysis (vortex        for 10 seconds if possible).    -   7. Place collected specimens into a double plastic bag first and        then into a STYROFOAM™ container (primary container) with Cool        Packs.        -   a. Consult with shipping company for the international            shipping process for necessary permits and documentations.    -   8. Make certain the requisition and sample list is placed in the        box, but on the outside of the double bag.    -   9. Securely seal the container and label with “Biological        Specimen.”

Example 7 Data Analysis for Quantitation of Signal Transduction PathwayProteins in Cancer Cells

This example illustrates the quantitation of the expression and/oractivation levels of one or more analytes such as one or more signaltransduction proteins in a biological sample (e.g., blood or tumortissue) against a standard curve generated for the particular analyte ofinterest.

In some embodiments, each CEER slide is scanned at three photomultiplier(PMT) gain settings to improve sensitivity and reduce the impact ofsaturation. Perkin Elmer ScanArray® Express software is used for spotfinding and signal quantitation. The identifiers for each spot areimported from a GenePix® Array List (.gal) file. The de-identified studyspecific number for each clinical sample on a slide is incorporated intothe resulting data set.

In other embodiments, background corrected signal intensities areaveraged for replicate spots printed in triplicate. The relativefluorescence value of the respective reagent blank is subtracted fromeach sample. Several quality criteria are used to filter data fromfurther analysis including limits on the spot footprint, coefficient ofvariation for spot replicates, overall pad background and the intensityof the reagent blank.

For each assay, a sigmoidal standard curve can be generated frommultiple (e.g., two, three, four, five, six, seven, etc.) concentrationsof serially diluted cell lysates prepared from cell lines such as MD-468(HER1 positive), SKBr3 (HER2 positive), BT474 (HER2 and p95HER2positive), HCC827 (c-MET and HER1 positive), T47D stimulated with IGF(IGF1R positive), and/or T47D stimulated with HRG (HER3 positive). Eachcurve can be plotted as a function of signal intensity vs. logconcentration derived units, CU (Computed Unit). The data can be fit toa five parameter equation (5PL) by nonlinear regression (Ritz, C. andStreibig, J. C., J Statistical Software, 12, 1-22 (2005)),simultaneously fitting all three dilutions of the capture antibody.Fitting is carried out using R, an open source statistical softwarepackage (Development Core Team, R: A language and environment forstatistical computing. R Foundation for Statistical Computing, Vienna,Austria. ISBN 3-900051-07-0(2008)). To avoid over parameterization ofthe mathematical model and thereby improve accuracy, four parameters canbe constrained, while each dilution can be solved for an individualinflection point. This process can be repeated for each PMT gain settingof 45, 50 and 60. This results in nine standard curves generated perassay, from three dilutions of capture antibody and three PMT scans. Thebuilt-in redundancy in the assay allows for one or more of thedilution/scan combinations to be eliminated if the fit of the standardcurve has an R² less than 0.95 and thus improves subsequent predictions.

CU Calculation (Based on Standard Curve)—The individual predictions fromeach of the standard curves (e.g., 3 capture antibody dilutions and 3PMT gain-set scanning) can be combined into a single, final prediction.For each prediction, the slope of the point on the standard curve iscalculated. This slope is taken with log-units on the x-axis, i.e., theunits in the denominator of the slope are log Computed Units (CU).Second, a weighted average of the predictions is calculated, where theweights are determined from the slopes. Specifically, the weights aresummed, and each point is given a weight equal to its slope divided bythe total slopes. Each assay can be validated against predictions forknown controls.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. Although the foregoing invention has beendescribed in some detail by way of illustration and example for purposesof clarity of understanding, it will be readily apparent to those ofordinary skill in the art in light of the teachings of this inventionthat certain changes and modifications may be made thereto withoutdeparting from the spirit or scope of the appended claims.

What is claimed is:
 1. A method for determining whether a human subject with a triple-negative breast cancer will respond to therapy with a combination of bevacizumab (Avastin®), carboplatin, and paclitaxel, the method comprising: (a) obtaining a breast tumor sample from the human subject; (b) determining an expression profile in said sample of VEGFR2 protein, c-KIT protein, HER1 protein, and IGF-1R protein using a proximity immunoassay by contacting the sample with (1) capture antibodies that bind to each of VEGFR2 protein, c-KIT protein, HER1 protein, and IGF-1R protein, and (2) detection antibodies comprising first and second activation state-independent antibodies that bind to each of VEGFR2 protein, c-KIT protein, HER1 protein, and IGF-1R protein, wherein the capture antibodies are attached to a solid support, wherein the first activation state-independent antibodies are labeled with glucose oxidase, wherein the second activation state-independent antibodies are labeled with horseradish peroxidase, and wherein the binding of the first and second activation state-independent antibodies in proximity to each other generates a detectable signal; (c) comparing said expression profile to a reference expression profile comprising a median expression level of VEGFR2 protein, c-KIT protein, HER1 protein, and IGF-1R protein in a human population of triple-negative breast tumors; and (d) determining whether the human subject with a triple-negative breast cancer will respond to therapy with a combination of bevacizumab (Avastin®), carboplatin, and paclitaxel based on altered expression of VEGFR2 protein, c-KIT protein, HER1 protein, and IGF-1R protein in the sample relative to the reference expression profile, wherein decreased expression of VEGFR2 protein relative to the median expression level of VEGFR2 protein, decreased expression of c-KIT protein relative to the median expression level of c-KIT protein, increased expression of HER1 protein relative to the median expression level of HER1 protein, and decreased expression of IGF-1R protein relative to the median expression level of IGF-1R protein is indicative of response to therapy with the combination of bevacizumab (Avastin®), carboplatin, and paclitaxel.
 2. The method of claim 1, wherein the triple-negative breast cancer is triple-negative metastatic breast cancer.
 3. The method of claim 1, wherein the paclitaxel is a nanoparticle albumin-bound (nab) paclitaxel. 